Cyclic ether vitamin D3 compounds, 1alpha(OH) 3-epi- vitamin D3 compounds and uses thereof

ABSTRACT

Novel cyclic ether vitamin D3 compounds having a cyclic ether side chain are disclosed. These compounds were first identified as metabolites of 3-epi vitamin D3 produced via a tissue-specific metabolic pathway which catalyzes the formation of a cyclic ether structure. Also disclosed are 1α(OH) 3-epi vitamin D3 compounds, which are produced via the epimerization of a 3-β-hydroxyl group of 1α(OH) vitamin D3 precursor in vivo. The vitamin D3 compounds of the present invention can be used as substitutes for natural and synthetic vitamin D3 compounds.

RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional applicationApplication No. 60/046, 690 filed on May 16, 1997, the contents of whichare incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] The importance of the vitamin D in the biological systems ofhigher animals has been recognized since its discovery by Mellanby in1920 (Mellanby, E. (1921) Spec. Rep. Ser. Med. Res. Council (GB) SRS61:4). It was in the interval of 1920-1930 that vitamin D officiallybecame classified as a “vitamin” that was essential for the normaldevelopment of the skeleton and maintenance of calcium and phosphoroushomeostasis.

[0003] Studies involving the metabolism of vitamin D₃ (cholecalciferol)were initiated with the discovery and chemical characterization of theplasma metabolite, 25-hydroxyvitamin D₃ [25(OH)D₃] (Blunt, J. W. et al.(1968) Biochemistry 6:3317-3322) and the hormonally active form,1α,25(OH)₂D₃ (Myrtle, J. F. et al. (1970) J. Biol. Chem. 245:1190-1196;Norman, A. W. et al. (1971) Science 173:51-54; Lawson, D. E. M. et al(1971) Nature 230:228-230; Holick, M. F. (1971) Proc. Natl. Acad. Sci.USA 68:803-804). The formulation of the concept of a vitamin D endocrinesystem was dependent both upon appreciation of the key role of thekidney in producing 1α, 25(OH)₂D₃ in a carefully regulated fashion(Fraser, D. R. and Kodicek, E (1970) Nature 288:764-766; Wong, R. G. etal. (1972) J. Clin. Invest. 51:1287-1291), and the discovery of anuclear receptor for 1α,25(OH)₂D₃ (VD₃R) in the intestine (Haussler, M.R. et al. (1969) Exp. Cell Res. 58:234-242; Tsai, H. C. and Norman, A.W. (1972) J. Biol. Chem. 248:5967-5975). The operation of the vitamin Dendocrine system depends on the following: first, on the presence ofcytochrome P450 enzymes in the liver (Bergman, T. and Postlind, H.(1991) Biochem. J. 276:427-432; Ohyama, Y and Okuda, K. (1991) J. Biol.Chem. 266:8690-8695) and kidney (Henry, H. L. and Norman, A. W. (1974)J. Biol. Chem. 249:7529-7535; Gray, R. W. and Ghazarian, J. G. (1989)Biochem. J. 259:561-568), and in a variety of other tissues to effectthe conversion of vitamin D₃ into biologically active metabolites suchas α,25(OH)₂D₃ and 24R,25(OH)₂D₃; second, on the existence of the plasmavitamin D binding protein (DBP) to effect the selective transport anddelivery of these hydrophobic molecules to the various tissue componentsof the vitamin D endocrine system (Van Baelen, H. et al. (1988) Ann NYAcad. Sci. 538:60-68; Cooke, N. E. and Haddad, J. G. (1989) Endocr. Rev.10:294-307; Bikle, D. D. et al. (1986) J. Clin. Endocrinol. Metab.63:954-959); and third, upon the existence of stereoselective receptorsin a wide variety of target tissues that interact with the agonist1α,25(OH)₂D₃ to generate the requisite specific biological responses forthis secosteroid hormone (Pike, J. W. (1991) Annu. Rev. Nutr.11:189-216). To date, there is evidence that nuclear receptors for1α,25(OH)₂D₃ (VD₃R) exist in more than 30 tissues and cancer cell lines(Reichel, H. and Norman, A. W. (1989) Annu. Rev. Med. 40:71-78).

[0004] Vitamin D₃ and its hormonally active forms are well-knownregulators of calcium and phosphorous homeostasis. These compounds areknown to stimulate, at least one of, intestinal absorption of calciumand phosphate, mobilization of bone mineral, and retention of calcium inthe kidneys. Furthermore, the discovery of the presence of specificvitamin D receptors in more than 30 tissues has led to theidentification of vitamin D₃ as a pluripotent regulator outside itsclassical role in calcium/bone homeostasis. A paracrine role for1α,25(OH)₂D₃ has been suggested by the combined presence of enzymescapable of oxidizing vitamin D₃ into its active forms, e.g.,25-OHD-1α-hydroxylase, and specific receptors in several tissues such asbone, keratinocytes, placenta, and immune cells. Moreover, vitamin D₃hormone and active metabolites have been found to be capable ofregulating cell proliferation and differentiation of both normal andmalignant cells (Reichel, H. et al. (1989) Ann. Rev. Med. 40:71-78).

[0005] Given the pluripotent activities of vitamin D₃ and itsmetabolites, much attention has focused on the development of syntheticanalogs of these compounds. However, clinical applications of vitamin D₃and its structural analogs have been limited by the undesired sideeffects elicited by these compounds after administration to a subject,such as the deregulation of calcium and phosphorous homeostasis in vivothat results in hypercalcemia.

SUMMARY OF THE INVENTION

[0006] The present invention is based, at least in part, on thediscovery of vitamin D3 compounds having a cyclic ether side chain,referred to hereinafter as “cyclic ether vitamin D3 compounds”, andwhich are represented by the formula I. This invention also describes3-epi forms of 1α-hydroxy-vitamin D3 compounds, which are represented bythe formula II. The cyclic ether and 1α-hydroxy-vitamin D3 compounds offormulas I and II, respectively, referred to hereinafter as “vitamin D3compounds of formulas I and II” can be produced in vivo via a pathwaywhich catalyzes the epimerization 3-β-hydroxy-vitamin D3 in certaintissues, e.g., keratinocytes, bone cells. The vitamin D3 compounds ofthe present invention can be used as substitutes for natural andsynthetic forms of vitamin D3.

[0007] Accordingly, the present invention pertains to cyclic ethervitamin D3 compounds having the formula (I) as follows:

[0008] , wherein A₁, A₂ and A₃ represent a single or a double bond; X,R₁, R₂, R₃, R₄ and R₅ can, e.g., be chosen individually from the groupof: a hydrogen, a halogen, a haloalkyl, a hydroxy, a hydroxy-protectinggroup, an alkyl, e.g., a lower alkyl, an alkenyl, an alkynyl, an alkoxy,an aryl group and a heterocyclic group. The orientation of the X groupcan be in either an α- or a β-configuration.

[0009] In a preferred embodiment, the cyclic ether vitamin D3 compoundis in its 3-epi configuration, wherein the orientation of the X group onthe A-ring is in an α-configuration.

[0010] The present invention also pertains to 3-epi forms of1α-hydroxy-vitamin D3 compounds having the formula II as follows:

[0011] , wherein A₁ represents a single, a double, e.g., a trans-double,a cis-double, or a triple bond; A₂, A₃ and A₄ represent a single or adouble bond; R₂, R₃, R₄, R₇, R₈ and R₉ can, e.g., be chosen individuallyfrom the group of: a hydrogen, a deuterium, a deuteroalkyl, a hydroxy,an alkyl, e.g., a lower alkyl, e.g., a C₁-C₄ alkyl, an alkoxide, anO-acyl, a halogen, e.g., a fluoride, a haloalkyl (e.g., a fluoroalkyl,—CF₃), a hydroxyalkyl, e.g., a hydroxyalkyl wherein the alkyl group is aC₄-C₁₀ alkyl, an amine or a thiol group, and wherein the pairs of R₂ andR₃, or R₄ and R₇ taken together can be an oxygen atom, e.g., as in acarbonyl moiety (

[0012] ); and R₅ and R₆ can, e.g., each be chosen individually from thegroup of: a hydrogen, a deuterium, a halogen, e.g., a fluoride, analkyl, e.g., a lower alkyl, e.g., a C₁-C₄ alkyl, a hydroxyalkyl, ahaloalkyl, e.g., a fluoroalkyl, and a deuteroalkyl. The amine or thiolgroup of R₂, R₃, R₄, R₇, R₈ and R₉ can be substituted to form, e.g., aprimary or a secondary amine, or a primary or secondary thiol, whereinthe substituents can be an alkyl or an aryl group, e.g., a substituenthaving 2- to 10-carbon atoms.

[0013] In another aspect, the present invention further pertains to apharmaceutical composition comprising, a therapeutically effectiveamount of a vitamin D3 compound having the formulas I or II, and apharmaceutically acceptable carrier.

[0014] In yet another aspect, this invention provides a method ofmodulating a biological activity of a vitamin D3-responsive cell. Thismethod comprising contacting the cell with an effective amount of anisolated vitamin D3 compound of formulas I and II such that modulationof the activity of the cell occurs.

[0015] Another aspect of the invention provides a method of treating ina subject, a disorder characterized by aberrant growth or activity of acell, comprising administering to the subject an effective amount of apharmaceutical composition of a vitamin D3 compound of formulas I and IIsuch that the growth or activity of the cell is reduced.

[0016] In a preferred embodiment, the vitamin D3 compound of formulas Iand II used in the treatment has improved biological properties comparedto vitamin D3, such as enhanced stability and/or reduced toxicity.

[0017] In one aspect, a method for inhibiting the proliferation and/oran inducing the differentiation of a hyperproliferative skin cell isprovided, wherein the hyperproliferative skin cell can be an epidermalcell or an epithelial cell. Accordingly, therapeutic methods fortreating hyperproliferative skin disorders, e.g., psoriasis, areprovided.

[0018] In certain embodiments, the instant method can be used for thetreatment of, or prophylactic prevention of a disorder characterized byaberrant cell growth of vitamin D3-responsive neoplastic cell, e.g., byadministering a pharmaceutical preparation of a vitamin D3 compoundhaving the formula as shown in I or II in an amount effective to inhibitgrowth of the neoplastic cells.

[0019] In another aspect, the subject method can be used to modulate animmune response, comprising administering to a subject a pharmaceuticalpreparation of a vitamin D compound so as to alter immune function inthe subject. In one embodiment, the method can be used in the treatmentof lymphoid cells, e.g., T cells, natural killer cells, so as tosuppress immune reactions, e.g., to decrease T cell activity, e.g., todecrease production of lymphokines such as IL-2 and IFN-γ, to decrease Tcell proliferation. In preferred embodients, the method can be used intreating graft rejection, autoimmunity and inflammation.

[0020] In yet another aspect, the vitamin D3 compound of the presentinvention are useful in the treatment of disorder characterized by aderegulation of calcium and phosphate metabolism, comprisingadministering to a subject a pharmaceutical preparation of a vitamin D3compounds of formulas I and II so as to ameliorate the deregulation incalcium and phosphate metabolism.

[0021] In a preferred embodiment, the disorder is osteoporosis. In otherembodiments, the vitamin D3 compounds of formulas I and II can be usedto treat diseases characterized by other deregulations in the metabolismof calcium and phosphate.

[0022] In another aspect, a method for inhibiting PTH secretion inparathyroid cell using the vitamin D3 compound of formulas I and II isprovided. Furthermore, therapeutic methods for treating secondaryhyperparathyroidism are also provided.

[0023] In yet another aspect, the present invention provides a method ofpreventing or protecting against neuronal loss by contacting a vitaminD3-responsive cell, e.g., a neuronal cell, with a vitamin D3 compound offormulas I and II to prevent or retard neuron loss.

[0024] In yet another aspect, the present invention provides a method ofmodulating the activity of a vascular smooth muscle cell by contacting avitamin D3-responsive smooth muscle cell with a vitamin D3 compound offormulas I and II to activate or, preferably, inhibit the activity ofthe cell.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1 is a compilation of the chemical structures of 266 vitaminD₃ compounds (Boullion, R. et al. (1995) Endocrinology Reviews 16(2):200-257, the contents of which including the figures depicted thereinare incorporated by reference). Each analog is identified by itschemical name and a one, two, or three-letter identification code.

[0026]FIG. 2 shows the HPLC profile and UV spectra of the metabolitesproduced in human keratinocytes incubated with 1α, 25(OH)₂-3-epi vitaminD₃.

[0027]FIG. 3 shows the mass spectra of 1α, 25(OH)₂-3-epi vitamin D₃ andits cyclic ether metabolite.

[0028]FIG. 4 shows the proposed metabolic pathway for the formation ofthe cyclic ether metabolite of 1α, 25(OH)₂-3-epi vitamin D₃.

[0029]FIG. 5A shows the metabolism of 1α(OH)-vitamin D₃ into its 3 epiform in the rat osteosarcoma cell line (UMR-106).

[0030]FIG. 5B is a schematic of the 3-epimerization of 1α(OH)D3 into1α(OH)-3-epi vitamin D₃.

[0031]FIG. 6 shows the mass spectra of 1α(OH)D₃ and its 3-epimetabolite.

[0032]FIG. 7 shows the HPLC profile and UV spectra of the metabolitesproduced in rat osteosarcoma cell lines (UMR-106) which were incubatedwith 1α(OH)D₃ for 24, 48, or 84 hours.

DETAILED DESCRIPTION OF THE INVENTION

[0033] The language “cyclic ether vitamin D3 compound” is intended toinclude all vitamin D3 compounds having a cyclic ether side chain,including 3-epimeric and non-3-epimeric of vitamin D3 as represented bythe general formula I.

[0034] As used herein, the terms “3-epi vitamin D3” or “3-epi D3”compounds are intended to include vitamin D3 compounds having asubstituent, e.g., a functional group, e.g., a hydroxyl group, attachedto the carbon at position 3 of the A-ring in an α-configuration ratherthan a β-configuration. The language “3-epi forms of 1α-hydroxy-vitaminD3 compounds” or “1α-hydroxy-3-epi-vitamin D3 compounds” is intended toinclude 1α-hydroxy-vitamin D3 compounds having the hydroxyl group,attached to the carbon at position 3 of the A-ring in an α-configurationrather than a β-configuration, and which are represented by the generalformula II as described in detail below.

[0035] The cyclic ether and 1α-hydroxy-vitamin D3 compounds of formulaeI and II, respectively, referred to hereinafter as “vitamin D3 compoundsof formulas I and II” can be produced in vivo via a pathway whichcatalyzes the epimerization 3-β-hydroxy-vitamin D3 in certain tissues,e.g., keratinocytes or bone cells.

[0036] The language “vitamin D3 compounds” or “cholecalciferols” (alsoreferred to herein as “D3 compounds”) is intended to include compoundswhich are structurally similar to vitamin D₃. Many of these compoundsare art-recognized and comprise a large number of natural precursors,metabolites, as well as synthetic analogs of the hormonally active1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃). This language is intended toinclude vitamin D₃, or an analog thereof, at any stage of itsmetabolism, as well as mixtures of different metabolic forms of vitaminD₃ or analogs thereof. Furthermore, the term “vitamin D₃ compound” alsoincludes synthetic analogs of vitamin D₃ illustrated in FIG. 1.

[0037] In the formulas presented herein, the various substituents areillustrated as joined to the steroid nucleus by one of these notations:a dotted line (----) indicating a substituent which is in theβ-orientation (i.e., above the plane of the ring), a wedged solid line (

) indicating a substituent which is in the α-orientation (i.e., belowthe plane of the molecule), or a solid line (—) indicating a substituentin the plane of the ring. It should be understood that thestereochemical convention in the steroid field is opposite from thegeneral chemical field, wherein a dotted line indicates a substituentwhich is in an α-orientation (i.e., below the plane of the molecule),and a wedged solid line indicates a substituent which is in theβ-orientation (i.e., above the plane of the ring). As shown, the A ringof the hormone 1α,25(OH)₂D₃ contains two asymetric centers at chiralcarbons-1 and −3, each one containing a hydroxyl group inwell-characterized configurations, namely the 1α- and 3β- hydroxylgroups.

[0038] Accordingly, the present invention pertains to cyclic ethervitamin D3 compounds having the formula (I) as follows:

[0039] , wherein A₁, A₂ and A₃ represent a single or a double bond; X,R₁, R₂, R₃, R₄ and R₅ can, e.g., be chosen individually from the groupof: a hydrogen, a halogen, a haloalkyl, a hydroxy, a hydroxy-protectinggroup, an alkyl, e.g., a lower alkyl, an alkenyl, an alkynyl, an alkoxy,an aryl group and a heterocyclic group. The orientation of the X groupcan be in either an α- or a β-configuration.

[0040] In a preferred embodiment, the cyclic ether vitamin D3 compoundis represented by the general formula I, wherein the orientation of theX group on the A-ring is in an α-configuration; A₁ is a single bond; A₂and A₃ are each a double bond; −X and R₁ are hydroxyl groups; R₂, R₃, R₄and R₅ are a hydrogen.

[0041] The present invention also pertains to 3-epi forms ofla-hydroxy-vitamin D3 compounds having the formula II:

[0042] , wherein A₁ represents a single, a double, e.g., a trans-double,a cis-double, or a triple bond; A₂, A₃ and A₄ represent a single or adouble bond; R₂, R₃, R₄, R₇, R₈ and R₉ can, e.g., be chosen individuallyfrom the group of: a hydrogen, a deuterium, a deuteroalkyl, a hydroxy,an alkyl, e.g., a lower alkyl, e.g., a C₁-C₄ alkyl, an alkoxide, anO-acyl, a halogen, e.g., a fluoride, a haloalkyl (e.g., a fluoroalkyl,—CF₃), a hydroxyalkyl, e.g., a hydroxyalkyl wherein the alkyl group is aC₄-C₁₀ alkyl, an amine or a thiol group, and wherein the pairs of R₂ andR₃, or R₄ and R₇ taken together can be an oxygen atom, e.g., as in acarbonyl moiety (

[0043] ); and R₅ and R₆ can, e.g., each be chosen individually from thegroup of: a hydrogen, a deuterium, a halogen, e.g., a fluoride, analkyl, e.g., a lower alkyl, e.g., a C₁-C₄ alkyl, a hydroxyalkyl, ahaloalkyl, e.g., a fluoroalkyl, and a deuteroalkyl. The amine or thiolgroup of R₂, R₃, R₄, R₇, R₈ and R₉ can be substituted to form, e.g., aprimary or a secondary amine, or a primary or a secondary thiol, whereinthe substituents can be an alkyl or an aryl group, e.g., a substituenthaving 2- to 10-carbon atoms.

[0044] In a preferred embodiment, A₁, A₂ and A₃ are each a single bond;A₄ is a double bond; R₂, R₃, R₅, R₆, R₈ and R₉ are each a hydrogen or analkyl, e.g., a methyl; and R₄ and R₇ are each a hydrogen, a hydroxy oran alkyl, e.g., a lower alkyl, e.g., a methyl or an ethyl group. Thechirality of the positions substituted by R₄ and R₇ can be in either anR- or an S-configuration.

[0045] Exemplary preferred 1α:-hydroxy vitamin D₃ compounds encompassedby formula II include: 1α hydroxy 3-epi vitamin D₃, 1α,24 dihydroxy3-epi vitamin D₃ (both 1α, 24R-dihydroxy 3-epi vitamin D₃ and 1α,24S-dihydroxy 3-epi vitamin D₃), 1α hydroxy 24-ethyl 3-epi vitamin D₃,1α: hydroxy 24-methyl 3-epi vitamin D₃ and 1α,24-dihydroxy 24-methyl3-epi vitamin D3 having the following chemical formulae:

[0046] A representation of 1α-hydroxy-vitamin D3 prior to 3-epiconversion is also depicted as analog BP in FIG. 1.

[0047] In yet another embodiment, the present invention providesisolated vitamin D3 compounds of formulae I and II, having at least onebiological activity of vitamin D3, and having improved biologicalproperties compared to vitamin D3, such as enhanced stability in vivoand/or reduced toxicity.

[0048] The term “epimer” or “epi” compounds is intended to includecompounds having a chiral carbon that varies in the orientation of asingle bond to a substituent on that carbon compared to thenaturally-occurring (or reference) compound, for example, a carbon wherethe orientation of the bond to the substituent is in an a-configuration,instead of a β-configuration. The 3-epimer forms of vitamin D3 compoundshaving the general formulas I and II have a substituent, e.g., ahydroxyl group, attached to the carbon at position 3 of the A-ring in anα-configuration rather than a β-configuration, whereas all othersubstituents can be in either an α- or a β-configuration.

[0049] As used herein, the term “substituent” refers to a moiety, forexample a functional group, attached to the carbon position 3 of the Aring of the vitamin D₃ compound that allows the compound to perform itsintended function. Accordingly, the term “substituent” is intended toinclude hydrogen, halogen, haloalkyl, hydroxy, hydroxy-protecting group,alkyl, e.g. lower alkyl, alkenyl, e.g., lower alkenyl, alkynyl, e.g.,lower alkynyl, alkoxy, aryl group and heterocyclic group.

[0050] The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner. The term “stereoisomers” or “isomers” refer to compoundswhich have identical chemical constitution, but differ with regard tothe arrangement of the atoms or groups in space. In particular,“enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another. An equimolar mixture oftwo enantiomers is called a “racemic mixture” or a “racemate”.“Diastereomers” refer to stereoisomers with two or more centers ofdissymmetry and whose molecules are not mirror images of one another.With respect to the nomenclature of a chiral center, terms “d” and “l”configuration are as defined by the IUPAC Recommendations. As to the useof the terms, diastereomer, racemate, epimer and enantiomer will be usedin their normal context to describe the stereochemistry of preparations.

[0051] As used herein, the language “isomeric counterparts of vitaminD3” or “non-epimeric forms” refers to stereoisomers of the 3-epi vitaminD3 compounds. For example, vitamin D3 compounds which have theorientation of the 3-hydroxy group in a β-configuration.

[0052] The terms “isolated” or “substantially purified” as usedinterchangeably herein refer to vitamin D₃ compounds in a non-naturallyoccurring state. The compounds can be substantially free of cellularmaterial or culture medium when naturally produced, or chemicalprecursors or other chemicals when chemically synthesized. In otherpreferred embodiments, the terms “isolated” or “substantially purified”also refer to preparations of a chiral compound which substantially lackone of the enantiomers, i.e., enantiomerically enriched or non-racemicpreparations of a molecule. Similarly, isolated epimers or diasteromersrefers to preparations of chiral compounds which are substantially freeof other stereochemical forms. For instance, isolated or substantiallypurified vitamin D₃ compounds includes synthetic or natural preparationsof a vitamin D₃ enriched for the stereoisomers having a substituentattached to the chiral carbon at position 3 of the A-ring in anα-configuration, and thus substantially lacking other isomers having aβ-configuration. Unless otherwise specified, such terms refer to vitaminD₃ compositions in which the ratio of α to β forms is greater that 1:1by weight. For instance, an isolated preparation of an a epimer means apreparation having greater than 50% by weight of the α-epimer relativeto the β stereoisomer, more preferably at least 75% by weight, and evenmore preferably at least 85% by weight. Of course the enrichment can bemuch greater than 85%, providing a “substantially epimer enriched”,which refers to preparations of a compound which have greater than 90%of the α-epimer relative to the β stereoisomer, and even more preferablygreater than 95%. The term “substantially free of the β stereoisomer”will be understood to have similar purity ranges.

[0053] As used herein, the language “alkyl” is art-recognized andincludes to the radical of saturated aliphatic groups, includingstraight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl(alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkylsubstituted alkyl groups. In preferred embodiments, a straight chain orbranched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g.,C₁-C₃₀ for straight chain, C₃-C₃₀ for branched chain), and morepreferably 20 or fewer. Likewise, preferred cycloalkyls have from 4-10carbon atoms in their ring structure, and more preferably have 5, 6 or 7carbons in the ring structure.

[0054] Unless the number of carbons is otherwise specified, “loweralkyl” as used herein means an alkyl group, as defined above, but havingfrom one to ten carbons, more preferably from one to six, and mostpreferably from one to four carbon atoms in its backbone structure,which may be straight or branched-chain, which may be straight orbranched-chain. Examples of lower alkyl groups include methyl, ethyl,n-propyl, i-propyl, tert.-butyl, hexyl, heptyl, octyl and so forth.Likewise, “lower alkenyl” and “lower alkynyl” have similar chainlengths. Preferred alkyl groups include lower alkyls. Examples ofalkylene groups are methylene, ethylene, propylene and so forth.

[0055] Moreover, the term alkyl as herein is intended to include both“unsubstituted alkyls” and “substituted alkyls”, the latter of whichrefers to alkyl moieties having substituents replacing a hydrogen on oneor more carbons of the hydrocarbon backbone. Such substituents caninclude, for example, halogen, hydroxyl, carbonyl (including aldehydes,ketones, carboxylates, and esters), alkoxyl, ether, phosphoryl, cyano,amino, acylamino, amido, amidino, imino, sulfhydryl, alkylthio,arylthio, thiolcarbonyl (including thiolformates, thiolcarboxylic acids,and thiolesters), sulfonyl, nitro, heterocyclyl, aralkyl, or an aromaticor heteroaromatic moiety. It will be understood by those skilled in theart that the moieties substituted on the hydrocarbon chain canthemselves be substituted, if appropriate. For instance, thesubstituents of a substituted alkyl may include substituted andunsubstituted forms of amino, acylaminos, iminos, amidos, phosphoryls(including phosphonates and phosphinates), sulfonyls (includingsulfates, sulfonatos, sulfarnoyls, and sulfonamidos), and silyl groups,as well as ethers, alkylthios, arylthios, carbonyls (including ketones,aldehydes, carboxylates, and esters), —CF₃, —CN and the like. Exemplarysubstituted alkyls are described below. Cycloalkyls can be furthersubstituted with alkyls, alkenyls, alkoxys, alkylthios, arylthios,aminoalkyls, carbonyl-substituted alkyls, —CF₃, cyano (—CN), and thelike.

[0056] The term “aralkyl”, as used herein, refers to an alkyl groupsubstituted with an aryl group (e.g., an aromatic or heteroaromaticgroup).

[0057] The terms “alkenyl” and “alkynyl” are art-recognized and includeto unsaturated aliphatic groups analogous in length and possiblesubstitution to the alkyls described above, but that contain at leastone double or triple bond respectively.

[0058] The terms “alkoxyl” is art-recognized and includes to an grouprepresented by the formula —O-alkyl. Representative alkoxyl groupsinclude methoxy, ethoxy, propoxy, tert-butoxy and the like. Unlessotherwise specified, an “alkoxy” group can be replaced with a grouprepresented by —O-alkenyl, —O-alkynyl, —O-aryl (i.e., an aryloxy group),or —O—heterocyclyl. An “ether” is two substituted or unsubstitutedhydrocarbons covalently linked by an oxygen. Accordingly, thesubstituent of, e.g., an alkyl that renders that alkyl an ether is orresembles an alkoxyl, such as can be represented by one of —O-alkyl,—O—alkenyl, —O-alkynyl, —O-aryl, or —O-heterocyclyl. The term “loweralkoxy” includes a lower alkyl group attached to the remainder of themolecule by oxygen.

[0059] Examples of alkoxy groups include methoxy, ethoxy, isopropoxy,tert.-butoxy and so forth. The term “phenyl alkoxy” refer to an alkoxygroup which is substituted by a phenyl ring. Examples of phenyl alkoxygroups are benzyloxy, 2-phenylethoxy, 4-phenylbutoxy and so forth. Theterm “alkanoyloxy group” refers to the residue of an alkylcarboxylicacid formed by removal of the hydrogen from the hydroxyl portion of thecarboxyl group. Examples of alkanoyloxy groups include formyloxy,acetoxy, butyryloxy, hexanolyoxy and so forth. The term “substituted” asapplied to “phenyl” refers to phenyl which is substituted with one ormore of the following groups: alkyl, halogen (i.e., fluorine, chlorine,bromine or iodine), nitro, cyano, trifluoromethly and so forth. The“alkanol” or a “hydroxyalkyl” refer to a compound derived by protonationof the oxygen atom of an alkoxy group. Examples of alkanols includemethanol, ethanol, 2-propanol, 2-methyl-2-propanol and the like.

[0060] As used herein the term “hydroxy-protecting group” includes anygroup commonly used for the protection of hydroxy functions duringsubsequent reactions, including, for example, acyl or alkylsilyl groupssuch as trimethylsilyl, triethylsilyl, t-butyldimethylsilyl andanalogous alkylated silyl radicals, or alkoxyalkyl groups such asmethoxymethyl, ethoxymethyl, methoxyethoxymethyl, tetrahydrofuranyl ortetrahydropyranyl. A “protected-hydroxy” is a hydroxy functionderivatized by one of the above hydroxy-protecting groupings.

[0061] As used herein, the term “halogen” designates —F, —Cl, —Br or —I;the term “sulfhydryl” or “thiol” means —SH; the term “hydroxyl” means—OH.

[0062] The term “aryl” is art-recognized and includes 5- and 6-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Aryl groups also include polycyclic fusedaromatic groups such as naphthyl, quinolyl, indolyl, and the like. Thosearyl groups having heteroatoms in the ring structure may also bereferred to as “aryl heterocycles”, “heteroaryls” or “heteroaromatics”.The aromatic ring can be substituted at one or more ring positions withsuch substituents as described above, as for example, halogen, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, acylamino,azido, nitro, sulfhydryl, imino, amido, amidino, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, arylthio,sulfonyl, sulfonamido, sulfamoyl, ketone, aldehyde, ester, aheterocyclyl, an aromatic or heteroaromatic moiety, —CF₃, —CN, or thelike. Aryl groups can also be fused or bridged with alicyclic orheterocyclic rings which are not aromatic so as to form a polycycle(e.g., tetralin).

[0063] The terms “heterocyclyl” or “heterocyclic group” areart-recognized and include 3- to 10-membered ring structures, morepreferably 4- to 7-membered rings, which ring structures include one tofour heteroatoms. Heterocyclyl groups include pyrrolidine, oxolane,thiolane, imidazole, oxazole, piperidine, piperazine, morpholine,lactones, lactams such as azetidinones and pyrrolidinones, lactones,sultams, sultones, and the like. The heterocyclic ring can besubstituted at one or more positions with such substituents as describedabove, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl,cycloalkyl, hydroxyl, amino, acylamino, nitro, sulfhydryl, imino, amido,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,arylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromaticor heteroaromatic moiety, —CF₃, —CN, or the like.

[0064] The terms “polycyclyl” or “polycyclic group” are art-recognizedand include two or more cyclic rings (e.g., cycloalkyls, cycloalkenyls,cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbonsare common to two adjoining rings, e.g., the rings are “fused rings”.Rings that are joined through non-adjacent atoms are termed “bridged”rings. Each of the rings of the polycycle can be substituted with suchsubstituents as described above, as for example, halogen, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, acylamino,nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl,carboxyl, silyl, ether, alkylthio, arylthio, sulfonyl, ketone, aldehyde,ester, a heterocyclyl, an aromatic or heteroaromatic moiety, —CF3, —CN,or the like.

[0065] Vitamin D Synthesis

[0066] The vitamin D3 compounds of the present invention can be preparedusing a variety of synthetic methods, as are known in the art. Forexample, many of the above-described compounds can be prepared bychemical synthesis, or alternatively by enzymatic conversion of a3β-vitamin D3 precursor, e.g., by perfusing a 3β-vitamin D3 precursor, avitamin D3 compound having the orientation of the hydroxy group atposition 3 of the A-ring in a β-configuration, in a tissue-containing anenzyme which catalyzes the epimerization of the 3-β-hydroxyl group tothe 3α form vitamin D3 compounds, e.g., keratinocytes or bone cells asdescribed in Examples I, II and IV.

[0067] For example, methods for synthesizing vitamin D3 compounds offormulas I and II are well known in the art (see e.g., Bouillon, R. etal., Endocrine Reviews 16(2):201-204; Ikekawa N. (1987) Med. Res. Rev.7:333-366; DeLuca H. F. and Ostrem V. K. (1988) Prog. Clin. Biol. Res.259:41-55; Ikekawa N. and Ishizuka S. (1992) CRC Press 8:293-316;Calverley M. J. and Jones G. (1992) Academic Press 193-270; Pardo R. andSantelli M. (1985) Bull Soc. Chim. Fr:98-114; Bythgoe B. (1980) Chem.Soc. Rev. 449-475; Quinkert G. (1985) Synform 3:41-122; Quinkert G.(1986) Synform 4:131-256; Quinkert G. (1987) Synform 5:1-85; Mathieu C.et al. (1994) Diabetologia 37:552-558; Dai H. and Posner G. H. (1994)Synthesis 1383-1398). Exemplary methods of synthesis include thephotochemical ring opening of a 1-hydroxylated side chain-modifiedderivative of 7-dehydrocholesterol which initially produces a previtaminthat is easily thermolyzed to vitamin D3 in a well known fashion (BartonD. H. R. et al. (1973) J. Am. Chem. Soc. 95:2748-2749; Barton D. H. R.(1974) JCS Chem. Comm. 203-204); phosphine oxide coupling methoddeveloped by (Lythgoe et al (1978) JCS Perkin Trans. 1:590-595) whichcomprises coupling a phosphine oxide to a Grundmann's ketone derivativeto directly produce a 1α,25(OH)₂D3 skeleton as described in BaggioliniE. G. et al. (1986) J. Org. Chem. 51:3098-3108; DeSchrijver J. andDeClercq P. J. (1993) Tetrahed Lett 34:4369-4372; Posner G. H and KinterC. M. (1990) J. Org. Chem. 55:3967-3969; semihydrogenation of dienynesto a previtamin structure that undergoes rearrangement to thecorresponding vitamin D3 analog as described by Harrison R. G. et al.(1974) JCS Perkin Trans. 1:2654-2657; Castedo L. et al. (1988) TetrahedLett 29:1203-1206; Mascarenas J. S. (1991) Tetrahedron 47:3485-3498;Barrack S. A. et al. (1988) J. Org. Chem. 53:1790-1796) and Okamura W.H. et al. (1989) J. Org. Chem. 54:4072-4083; the vinylallene approachinvolving intermediates that are subsequently arranged using heat or acombination of metal catalyzed isomerization followed by sensitizedphotoisomerization (Okamura W. H. et al. (1989) J. Org. Chem.54:4072-4083; Van Alstyne E. M. et al. (1994) J. Am. Chem. Soc.116:6207-6210); the method described by Trost et al. B. M. et al. J. Am.Chem. Soc. 114:9836-9845; Nagasawa K. et al. (1991) Tetrahed Lett32:4937-4940 involves an acyclic A-ring precursor which isintramolecular cross-coupled to the bromoenyne leading directly to theformation of 1,25(OH)₂D3 skeleton; a tosylated derivative which isisomerized to the i-steroid that can be modified at carbon-1 and thensubsequently back-isomerized under sovolytic conditions to formα,25(OH)₂D2 or analogs thereof (Sheves M. and Mazur Y. (1974) J. Am.Chem. Soc. 97:6249-6250; Paaren H. E. et al. (1980) J. Org. Chem.45:3253-3258; Kabat M. et al. (1991) Tetrahed Lett 32:2343-2346; WilsonS. R. et al. (1991) Tetrahed Lett 32:2339-2342); the direct modificationof vitamin D derivatives to 1-oxygenated 5, 6-trans vitamin D asdescribed in (Andrews D. R. et al. (1986) J. Org. Chem. 51:1635-1637);the Diels-Alders cycloadduct method of previtamin D3 can be used tocyclorevert to 1α,25(OH)₂D2 through the intermediary of a previtaminform via thermal isomerization (Vanmaele L. et al. (1985) Tetrahedron41:141-144); and, a final method entails the direct modification of1α,25(OH)₂D2 or an analog through use of suitable protecting groups suchas transition metal derivatives or by other chemical transformations(Okarmura W. H. et al. (1992) J. Cell Biochem. 49:10-18). Additionalmethods for synthesizing vitamins D2 compounds are described in, forexample, Japanese Patent Disclosures Nos. 62750/73, 26858/76, 26859/76,and 71456/77; U.S. Pat. Nos. 3,639,596; 3,715,374; 3,847,955 and3,739,001.

[0068] Examples of the compounds of this invention having a saturatedside chain can be prepared according to the general process illustratedand described in U.S. Pat. No. 4,927,815, the description of which isincorporated herein by reference. Examples of the compounds of thisinvention having an unsaturated side chain is can be prepared accordingto the general process illustrated and described in U.S. Pat. No.4,847,012, the description of which is incorporated herein by reference.Examples of the compounds of this invention wherein R groups togetherrepresent a cyclopentano group can be prepared according to the generalprocess illustrated and described in U.S. Pat. No. 4,851,401, thedescription of which incorporated herein by reference.

[0069] Another synthetic strategy for the preparation ofside-chain-modified analogues of 1α,25-dihydroxyergocalciferol isdisclosed in Kutner et al., The Journal of Organic Chemistry, 1988,53:3450-3457. In addition, the preparation of 24-homo and 26-homovitamin D analogs are disclosed in U.S. Pat. No. 4,717,721, thedescription of which is incorporated herein by reference.

[0070] The enantioselective synthesis of chiral molecules is now stateof the art. Through combinations of enantioselective synthesis andpurification techniques, many chiral molecules can be synthesized as anenantiomerically enriched preparation. For example, methods have beenreported for the enantioselective synthesis of A-ring diastereomers of1α,25(OH)₂D3 as described in Muralidharan et al. (1993) J. Organic Chem.58(7): 1895-1899 and Norman et al. (1993) J. Biol. Chem. 268(27):20022-30. Other methods for the enantiomeric synthesis of variouscompounds known in the art include, inter alia, epoxides (see, e.g.,Johnson, R. A.; Sharpless, K. B. In Catalytic Asymmetric Synthesis;Ojima, I., Ed.: VCH: New York, 1993; Chapter 4.1. Jacobsen, E. N. Ibid.Chapter 4.2), diols (e.g., by the method of Sharpless, J. Org. Chem.(1992) 57:2768), and alcohols (e.g., by reduction of ketones, E. J.Corey et al., J. Am. Chem. Soc. (1987) 109:5551). Other reactions usefulfor generating optically enriched products include hydrogenation ofolefins (e.g., M. Kitamura et al., J. Org. Chem. (1988) 53:708);Diels-Alder reactions (e.g., K. Narasaka et al., J. Am. Chem. Soc.(1989) 111:5340); aldol reactions and alkylation of enolates (see, e.g.,D. A. Evans et al., J. Am. Chem. Soc. (1981) 103:2127; D. A. Evans etal., J. Am. Chem. Soc. (1982) 104:1737); carbonyl additions (e.g., R.Noyori, Angew. Chem. Int. Ed. Eng. (1991) 30:49); and ring-opening ofmeso-epoxides (e.g., Martinez, L. E.; Leighton J. L., Carsten, D. H.;Jacobsen, E. N. J. Am. Chem. Soc. (1995) 117:5897-5898). The use ofenymes to produce optically enriched products is also well known in theart (e.g., M. P. Scheider, ed. “Enzymes as Catalysts in OrganicSynthesis”, D. Reidel, Dordrecht (1986).

[0071] Chiral synthesis can result in products of high stereoisomerpurity. However, in some cases, the stereoisomer purity of the productis not sufficiently high. The skilled artisan will appreciate that theseparation methods described herein can be used to further enhance thestereoisomer purity of the vitamin D3-epimer obtained by chiralsynthesis.

[0072] Separation of isomers can be accomplished in several ways knownin the art. An exemplary straight phase and reverse phase HPLC systemused to separate natural or synthetic diastereomers of 1α,25(OH)₂D3 isdetailed in the appended example and illustrated in FIG. 2. Furthermethods for separating a racemic mixture of two enantiomers includechromatography using a chiral stationary phase (see, e.g., “ChiralLiquid Chromatography”, W. J. Lough, Ed. Chapman and Hall, New York(1989)). Enantiomers can also be separated by classical resolutiontechniques. For example, formation of diastereomeric salts andfractional crystallization can be used to separate enantiomers. For theseparation of enantiomers of carboxylic acids, the diastereomeric saltscan be formed by addition of enantiomerically pure chiral bases such asbrucine, quinine, ephedrine, strychnine, and the like. Alternatively,diastereomeric esters can be formed with enantiomerically pure chiralalcohols such as menthol, followed by separation of the diastereomericesters and hydrolysis to yield the free, enantiomerically enrichedcarboxylic acid. For separation of the optical isomers of aminocompounds, addition of chiral carboxylic or sulfonic acids, such ascamphorsulfonic acid, tartaric acid, mandelic acid, or lactic acid canresult in formation of the diastereomeric salts.

[0073] Pharmaceutical Compositions

[0074] In another aspect, the present invention providespharmaceutically acceptable compositions which comprise atherapeutically-effective amount of one or more of the isolated vitaminD₃ compounds of formulas I and II, formulated together with one or morepharmaceutically acceptable carrier(s).

[0075] In a preferred embodiment, these pharmaceutical compositions aresuitable for topical or oral administration to a subject. In otherembodiments, as described in detail below, the pharmaceuticalcompositions of the present invention may be specially formulated foradministration in solid or liquid form, including those adapted for thefollowing: (1) oral administration, for example, drenches (aqueous ornon-aqueous solutions or suspensions), tablets, boluses, powders,granules, pastes; (2) parenteral administration, for example, bysubcutaneous, intramuscular or intravenous injection as, for example, asterile solution or suspension; (3) topical application, for example, asa cream, ointment or spray applied to the skin; (4) intravaginally orintrarectally, for example, as a pessary, cream or foam; or (5) aerosol,for example, as an aqueous aerosol, liposomal preparation or solidparticles containing the compound.

[0076] In certain embodiments, the subject is a mammal, e.g., a primate,e.g., a human. As used herein, the language “subject” is intended toinclude human and non-human animals. Preferred human animals include ahuman patient having a disorder characterized by the aberrant activityof a vitamin D₃-responsive cell. The term “non-human animals” of theinvention includes all vertebrates, e.g., mammals and non-mammals, suchas non-human primates, sheep, dog, cow, chickens, amphibians, reptiles,etc.

[0077] The phrase “therapeutically-effective amount” as used hereinmeans that amount of a vitamin D₃ compound(s) of formulas I and II, orcomposition comprising such a compound which is effective for thecompound to produce its intended function, e.g., the modulation ofactivity of a vitamin D₃-response cell. The effective amount can varydepending on such factors as the type of cell growth being treated orinhibited, the particular type of vitamin D₃ compound, the size of thesubject, or the severity of the undesirable cell growth or activity. Oneof ordinary skill in the art would be able to study the aforementionedfactors and make the determination regarding the effective amount of thevitamin D₃ compound of formulas I and II without undue experimentation.

[0078] In certain embodiments, one or more vitamin D₃ compounds asrepresented by formulas I and II may be administered alone, or as partof combinatorial therapy. For example, the vitamin D₃ compounds can beconjointly administered with one or more agents such as mitoticinhibitors, alkylating agents, antimetabolites, nucleic acid,intercalating agents, topoisomerase inhibitors, agents which promoteapoptosis, and/or agents which modulate immune responses. The effectiveamount of vitamin D₃ compound used can be modified according to theconcentrations of the other agents used.

[0079] In vitro assay using keratinocytes or parathyroid cells, or anassay similar thereto (e.g., differing in choice of cells, e.g., bonecells, intestinal cells, neoplastic cells) can be used to determine an“effective amount” of the vitamin D₃ compounds of formulas I and II, orcombinations thereof. The ordinarily skilled artisan would select anappropriate amount of each individual compound in the combination foruse in the aforementioned in vitro assay or similar assays. Changes incell activity or cell proliferation can be used to determine whether theselected amounts are “effective amount” for the particular combinationof compounds. The regimen of administration also can affect whatconstitutes an effective amount. As described in detail below, vitaminD₃ compounds of formulas I and II can be administered to the subjectprior to, simultaneously with, or after the administration of the otheragent(s). Further, several divided dosages, as well as staggereddosages, can be administered daily or sequentially, or the dose can beproportionally increased or decreased as indicated by the exigencies ofthe therapeutic situation.

[0080] The phrase “pharmaceutically acceptable” is employed herein torefer to those vitamin D₃ compounds of formulas I and II, compositionscontaining such compounds, and/or dosage forms which are, within thescope of sound medical judgment, suitable for use in contact with thetissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

[0081] The phrase “pharmaceutically-acceptable carrier” as used hereinmeans a pharmaceutically-acceptable material, composition or vehicle,such as a liquid or solid filler, diluent, excipient, solvent orencapsulating material, involved in carrying or transporting the subjectchemical from one organ, or portion of the body, to another organ, orportion of the body. Each carrier must be “acceptable” in the sense ofbeing compatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (1₃) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

[0082] Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

[0083] Examples of pharmaceutically-acceptable antioxidants include: (1)water soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfiteand the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metalchelating agents, such as citric acid, ethylenediamine tetraacetic acid(EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0084] Compositions containing the vitamin D₃ compounds of the presentinvention include those suitable for oral, nasal, topical (includingbuccal and sublingual), rectal, vaginal, aerosol and/or parenteraladministration. The compositions may conveniently be presented in unitdosage form and may be prepared by any methods well known in the art ofpharmacy. The amount of active ingredient which can be combined with acarrier material to produce a single dosage form will vary dependingupon the host being treated, the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the compound which produces a therapeutic effect. Generally, out ofone hundred percent, this amount will range from about 1 percent toabout ninety-nine percent of active ingredient, preferably from about 5percent to about 70 percent, most preferably from about 10 percent toabout 30 percent.

[0085] Methods of preparing these compositions include the step ofbringing into association a vitamin D₃ compound(s) of formulas I and IIwith the carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a vitamin D₃ compound with liquid carriers, orfinely divided solid carriers, or both, and then, if necessary, shapingthe product.

[0086] Compositions of the invention suitable for oral administrationmay be in the form of capsules, cachets, pills, tablets, lozenges (usinga flavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a vitamin D₃ compound(s)of formulas I and II as an active ingredient. A compound may also beadministered as a bolus, electuary or paste.

[0087] In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically-acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as,for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, acetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols andthe like.

[0088] A tablet may be made by compression or molding, optionally withone or more accessory ingredients. Compressed tablets may be preparedusing binder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered peptide orpeptidomimetic moistened with an inert liquid diluent.

[0089] The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

[0090] Liquid dosage forms for oral administration of the vitamin D₃compound(s) of the invention include pharmaceutically acceptableemulsions, microemulsions, solutions, suspensions, syrups and elixirs.In addition to the active ingredient, the liquid dosage forms maycontain inert diluents commonly used in the art, such as, for example,water or other solvents, solubilizing agents and emulsifiers, such asethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof.

[0091] Besides inert diluents, the oral compositions can also includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, coloring, perfuming and preservative agents.

[0092] Suspensions, in addition to the active vitamin D₃ compound(s) maycontain suspending agents as, for example, ethoxylated isostearylalcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

[0093] Pharmaceutical compositions of the invention for rectal orvaginal administration may be presented as a suppository, which may beprepared by mixing one or more vitamin D₃ compound(s) of formulas I andII with one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active agent.

[0094] Compositions of the present invention which are suitable forvaginal administration also include pessaries, tampons, creams, gels,pastes, foams or spray formulations containing such carriers as areknown in the art to be appropriate.

[0095] Dosage forms for the topical or transdermal administration of avitamin D₃ compound(s) of formulas I and II include powders, sprays,ointments, pastes, creams, lotions, gels, solutions, patches andinhalants. The active vitamin D₃ compound may be mixed under sterileconditions with a pharmaceutically-acceptable carrier, and with anypreservatives, buffers, or propellants which may be required.

[0096] The ointments, pastes, creams and gels may contain, in additionto vitamin D₃ compound(s) of formulas I and II, excipients, such asanimal and vegetable fats, oils, waxes, paraffins, starch, tragacanth,cellulose derivatives, polyethylene glycols, silicones, bentonites,silicic acid, talc and zinc oxide, or mixtures thereof.

[0097] Powders and sprays can contain, in addition to a vitamin D₃compound(s) of formulas I and II, excipients such as lactose, talc,silicic acid, aluminum hydroxide, calcium silicates and polyamidepowder, or mixtures of these substances. Sprays can additionally containcustomary propellants, such as chlorofluorohydrocarbons and volatileunsubstituted hydrocarbons, such as butane and propane.

[0098] The vitamin D₃ compound(s) of formulas I and II can bealternatively administered by aerosol. This is accomplished by preparingan aqueous aerosol, liposomal preparation or solid particles containingthe compound. A nonaqueous (e.g., fluorocarbon propellant) suspensioncould be used. Sonic nebulizers are preferred because they minimizeexposing the agent to shear, which can result in degradation of thecompound.

[0099] Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the agent together with conventionalpharmaceutically acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular compound, buttypically include nonionic surfactants (Tweens, Pluronics, orpolyethylene glycol), innocuous proteins like serum albumin, sorbitanesters, oleic acid, lecithin, amino acids such as glycine, buffers,salts, sugars or sugar alcohols. Aerosols generally are prepared fromisotonic solutions.

[0100] Transdermal patches have the added advantage of providingcontrolled delivery of a vitamin D₃ compound(s) of formulas I and II tothe body. Such dosage forms can be made by dissolving or dispersing theagent in the proper medium. Absorption enhancers can also be used toincrease the flux of the peptidomimetic across the skin. The rate ofsuch flux can be controlled by either providing a rate controllingmembrane or dispersing the peptidomimetic in a polymer matrix or gel.

[0101] Ophthalmic formulations, eye ointments, powders, solutions andthe like, are also contemplated as being within the scope of thisinvention.

[0102] Pharmaceutical compositions of this invention suitable forparenteral administration comprise one or more vitamin D₃ compound(s) offormulas I and II in combination with one or morepharmaceutically-acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.

[0103] Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

[0104] These compositions may also contain adjuvants such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

[0105] In some cases, in order to prolong the effect of a drug, it isdesirable to slow the absorption of the drug from subcutaneous orintramuscular injection. This may be accomplished by the use of a liquidsuspension of crystalline or amorphous material having poor watersolubility. The rate of absorption of the drug then depends upon itsrate of dissolution which, in turn, may depend upon crystal size andcrystalline form. Alternatively, delayed absorption of aparenterally-administered drug form is accomplished by dissolving orsuspending the drug in an oil vehicle.

[0106] Injectable depot forms are made by forming microencapsulematrices of vitamin D₃ compound(s) of formulas I and II in biodegradablepolymers such as polylactide-polyglycolide. Depending on the ratio ofdrug to polymer, and the nature of the particular polymer employed, therate of drug release can be controlled. Examples of other biodegradablepolymers include poly(orthoesters) and poly(anhydrides). Depotinjectable formulations are also prepared by entrapping the drug inliposomes or microemulsions which are compatible with body tissue.

[0107] When the vitamin D₃ compound(s) of the present invention areadministered as pharmaceuticals, to humans and animals, they can begiven per se or as a pharmaceutical composition containing, for example,0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient incombination with a pharmaceutically acceptable carrier.

[0108] The term “administration,” is intended to include routes ofintroducing a subject the 3-epimer vitamin D₃ compound of formula I toperform their intended function. Examples of routes of administrationwhich can be used include injection (subcutaneous, intravenous,parenterally, intraperitoneally, intrathecal, etc.), oral, inhalation,rectal and transdermal. The pharmaceutical preparations are of coursegiven by forms suitable for each administration route. For example,these preparations are administered in tablets or capsule form, byinjection, inhalation, eye lotion, ointment, suppository, etc.administration by injection, infusion or inhalation; topical by lotionor ointment; and rectal by suppositories. Oral administration ispreferred. The injection can be bolus or can be continuous infusion.Depending on the route of administration, the vitamin D₃ compound offormulas I and II can be coated with or disposed in a selected materialto protect it from natural conditions which may detrimentally effect itsability to perform its intended function. The vitamin D₃ compound offormulas I and II can be administered alone, or in conjunction witheither another agent as described above or with a pharmaceuticallyacceptable carrier, or both. The vitamin D₃ compound can be administeredprior to the administration of the other agent, simultaneously with theagent, or after the administration of the agent. Furthermore, thevitamin D₃ compound of formulas I and II can also be administered in aproform which is converted into its active metabolite, or more activemetabolite in vivo.

[0109] The phrases “parenteral administration” and “administeredparenterally” as used herein means modes of administration other thanenteral and topical administration, usually by injection, and includes,without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticulare, subcapsular, subarachnoid, intraspinal and intrastemalinjection and infusion.

[0110] The phrases “systemic administration,” “administeredsystemically”, “peripheral administration” and “administeredperipherally” as used herein mean the administration of a vitamin D₃compound(s) of formulas I and II, such that it enters the patient'ssystem and, thus, is subject to metabolism and other like processes, forexample, subcutaneous administration.

[0111] These vitamin D₃ compound(s) of formulas I and II may beadministered to a “subject”, e.g., mammals, e.g., humans and otheranimals. Administration can be carried out by any suitable route ofadministration, including orally, nasally, as by, for example, a spray,rectally, intravaginally, parenterally, intracisternally and topically,as by powders, ointments or drops, including buccally and sublingually.

[0112] Regardless of the route of administration selected, the vitaminD₃ compound(s) of formulas I and II, which may be used in a suitablehydrated form, and/or the pharmaceutical compositions of the presentinvention, are formulated into pharmaceutically-acceptable dosage formsby conventional methods known to those of skill in the art.

[0113] Actual dosage levels and time course of administration of theactive ingredients in the pharmaceutical compositions of this inventionmay be varied so as to obtain an amount of the active ingredient whichis effective to achieve the desired therapeutic response for aparticular patient, composition, and mode of administration, withoutbeing toxic to the patient. Exemplary dose range is from 0.1 to 10 μgper day.

[0114] Uses of the Vitamin D Compounds of the Invention

[0115] Another aspect of the invention pertains to isolated vitamin D₃compounds of formulas I and II having at least one biological activityof vitamin D₃, and having improved biological properties whenadministered into a subject than vitamin D₃ under the same conditions,as well as, methods of testing and using these compounds to treatdisorders involving an aberrant activity of a vitamin D3-responsivecell, e.g., neoplastic cells, hyperproliferative skin cells, parathyroidcells, immune cells and bone cells, among others.

[0116] The language “biological activities” of vitamin D₃ is intended toinclude all activities elicited by vitamin D₃ compounds of formulas Iand II in a responsive cell. This term includes genomic and non-genomicactivities elicited by these compounds (Bouillon, R. et al. (1995)Endocrinology Reviews 16(2):206-207; Norman A. W. et al. (1992) J.Steroid Biochem Mol. Biol 41:231-240; Baran D. T. et al. (1991) J. BoneMiner Res. 6:1269-1275; Caffrey J. M. and Farach-Carson M. C. (1989) J.Biol. Chem. 264:20265-20274; Nemere 1. et al. (1984) Endocrinology115:1476-1483).

[0117] As used herein, the term “vitamin D3-responsive cell” includesany cell which is is capable of responding to a vitamin D₃ compoundhaving the formula I or II, and is associated with disorders involvingan aberrant activity of hyperproliferative skin cells, parathyroidcells, neoplastic cells, immune cells, and bone cells. These cells canrespond to vitamin D₃ activation by triggering genomic and/ornon-genomic responses that ultimately result in the modulation of cellproliferation, differentiation survival, and/or other cellularactivities such as hormone secretion. In a preferred embodiment, theultimate responses of a cell are inhibition of cell proliferation and/orinduction of differentiation-specific genes. Exemplary vitamin D₃responsive cells include immune cells, bone cells, neuronal cells,endocrine cells, neoplastic cells, epidermal cells, endodermal cells,smooth muscle cells, among others.

[0118] As used herein, the language “vitamin D₃ agonist” refers to acompound which potentiates, induces or otherwise enhances a biologicalactivity of vitamin D₃ in a responsive cell. In certain embodiments, anagonist may induce a genomic activity, e.g., activation of transcriptionby a vitamin D₃ nuclear receptor, or a non-genomic vitamin D₃ activity,e.g., potentiation of calcium channel activity. In other embodiments,the agonist potentiates the sensitivity of the receptor to anothervitamin D₃ compound, e.g., treatment with the agonist lowers theconcentration of vitamin D₃ compound required to induce a particularbiological response. The language “vitamin D₃ antagonist” is intended toinclude those compounds that oppose any biological activity of a vitaminD₃ compound.

[0119] The language “non-genomic” vitamin D₃ activities include cellular(e.g., calcium transport across a tissue) and subcellular activities(e.g., membrane calcium transport opening of voltage-gated calciumchannels, changes in intracellular second messengers) elicited byvitamin D₃ compounds in a responsive cell. Electrophysiological andbiochemical techniques for detecting these activities are known in theart. An example of a particular well-studied non-genomic activity is therapid hormonal stimulation of intestinal calcium mobilization, termed“transcaltachia” (Nemere 1. et al. (1984) Endocrinology 115:1476-1483;Lieberherr M. et al. (1989) J. Biol. Chem. 264:20403-20406; Wali R. K.et al. (1992) Endocrinology 131:1125-1133; Wali R. K. et al. (1992) Am.J. Physiol. 262:G945-G953; Wali R. K. et al. (1990) J. Clin. Invest.85:1296-1303; Bolt M. J. G. et al. (1993) Biochem. J. 292:271-276).Detailed descriptions of experimental transcaltachia are provided inNorman, A. W. (1993) Endocrinology 268(27):20022-20030; Yoshimoto, Y.and Norman, A. W. (1986) Endocrinology 118:2300-2304. Changes in calciumactivity and second messenger systems are well known in the art and areextensively reviewed in Bouillion, R. et al. (1995) Endocrinology Review16(2): 200-257; the description of which is incorporated herein byreference.

[0120] Exemplary systems and assays for testing non-genomic activity areextensively described in the following references, liver (Baran D. T. etal. (1989) FEBS Lett 259:205-208; Baran D. T. et al. (1990) J. BoneMiner Res. 5:517-524;; rat osteoblasts, e.g., ROS 17/2.8 cells (Baran D.T. et al. (1991) J. Bone Miner Res. 6:1269-1275; Caffrey J. M. (1989) J.Biol. Chem. 264:20265-20274; Ciyitelli R. et al. (1990) Endocrinology127:2253-2262), muscle (DeBoland A. R. and Boland R. L. (1993) Biochem.Biophys Acta Mol. Cell Res. 1179:93-104; Morelli S. et al. (1993)Biochem J. 289:675-679; Selles J. and Boland R. L. (1991) Mol. CellEndocrinol. 82:229-235), and in parathyroid cells (Bourdeau A. et al.(1990) Endocrinology 127:2738-2743).

[0121] The language “genomic” activities or effects of vitamin D₃ isintended to include those activities mediated by the nuclear/cytosolreceptor for 1α,25(OH)₂D₃ (VD3R), e.g., transcriptional activation oftarget genes. The term “VD3Rs” is intended to include members of thetype II class of steroid/thyroid superfamily of receptors (Stunnenberg,H. G. (1993) Bio Essays 15(5):309-15), which are able to bindtransactivate through the vitamin D response element (VDRE) in theabsence of a ligand (Damm et al. (1989) Nature 339:593-97; Sap et al.Nature 343:177-180). As used herein “VDREs” refer to a DNA sequencescomposed of half-sites arranged as direct repeats. It is known in theart that type II receptors do not bind to their respective binding siteas homodimers but require an auxiliary factor, RXR (e.g. RXRα, RXRβ,RXRγ) for high affinity binding Yu et al.(1991) Cell67:1251-1266; Buggeet al. (1992) EMBO J. 11:1409-1418; Kliewer et al. (1992) Nature355:446-449; Leid et al. (1992) EMBO J. 11:1419-1435; Zhang et al.(1992) Nature 355:441-446).

[0122] Following binding, the transcriptional activity of a target gene(i.e., a gene associated with the specific DNA sequence) is enhanced asa function of the ligand bound to the receptor heterodimer. Exemplaryvitamin D₃-responsive genes include osteocalcin, osteopontin,calbindins, parathyroid hormone (PTH), 24-hydroxylase, and α_(v)β₃-integrin. Genomic activities elicited by vitamin D3 compounds can betested by detecting the transcriptional upregulation of a vitamin D₃responsive gene in a cell containing VD3R_(S). For example, the steadystate levels of responsive gene mRNA or protein, e.g. calbindin gene,osteocalcin gene, can be detected in vivo or in vitro. Suitable cellsthat can be used include any vitamin D3-responsive cell, e.g.,keratinocytes, parathyroid cells, MG-63 cell line, among others.

[0123] In accordance with a still further embodiment of the presentinvention, convenient screening methods can be established in cell linescontaining VD₃R_(S), comprising (i) establishing a culture of thesecells which include a reporter gene construct having a reporter genewhich is expressed in an VD₃R-dependent fashion; (ii) contacting thesecells with vitamin D3 compounds of formulas I and II; and (iii)monitoring the amount of expression of the reporter gene. Expression ofthe reporter gene reflects transcriptional activity of the VD₃R_(S)protein. Typically, the reporter gene construct will include a reportergene in operative linkage with one or more transcriptional regulatoryelements responsive to VD₃R_(S), e.g., the VD₃R_(S) response element(VDRE) known in the art. The amount of transcription from the reportergene may be measured using any method known to those of skill in the artto be suitable. For example, specific mRNA expression may be detectedusing Northern blots or specific protein product may be identified by acharacteristic stain, immunoassay or an intrinsic activity. In preferredembodiments, the gene product of the reporter is detected by anintrinsic activity associated with that product. For instance, thereporter gene may encode a gene product that, by enzymatic activity,gives rise to a detection signal based on color, fluorescence, orluminescence. The amount of expression from the reporter gene is thencompared to the amount of expression in either the same cell in theabsence of the test compound or it may be compared with the amount oftranscription in a substantially identical cell that lacks the specificreceptors. Agonistic vitamin D₃ compounds can then be readily detectedby the increased activity or concentration of these reporter genesrelative to untransfected controls.

[0124] After identifying certain test compounds as potential agonists orantagonists of vitamin D₃ compounds, the practioner of the subject assaywill continue to test the efficacy and specificity of the selectedcompounds both in vitro and in vivo. Whether for subsequent in vivotesting, or for administration to an animal as an approved drug, agentsidentified in the subject assay can be formulated in pharmaceuticalpreparations, such as described above, for in vivo administration to ananimal, preferably a human.

[0125] As described herein, the vitamin D3 compounds of the presentinvention show improved biological properties than vitamin D3. As usedherein, the language “improved biological properties” refers to anyactivity inherent in a vitamin D3 compound of formula I or II thatenhances its effectiveness in vivo. In a preferred embodiment, this termrefers to any qualitative or quantitative improved therapeutic propertyof a vitamin D₃ compound, such as enhanced stability in vivo and/orreduced toxicity, e.g., reduced hypercalcemic activity. The improvedbiological property may occur in both a tissue-specific and non-specificmanner. For example, certain tissues may be capable of metabolizingvitamin D₃ into unique metabolites that enhance in a tissue-specificmanner the biological activities of this compound.

[0126] The increased stability of the vitamin D3 compounds of formulas Iand II can be demonstated in incubation studies, wherein a significantlyhigher concentration of the such vitamin D3 after prolonged incubationsin vivo or in vitro, or an increase in the binding to plasma vitamin Dbinding protein (DBP) compared to vitamin D3 indicates a compound havingenhanced stability (See A. W. Norman et al. J. Biol. Chem. 268 (27):20022-20030).

[0127] The language “reduced toxicity” is intended to include areduction in any undesired side effect elicited by a vitamin D3 compoundof formula I or II when administered in vivo, e.g., a reduction in thehypercalcemic activity. The language “hypercalcemia” or “hypercalcemicactivity” is intended to have its accepted clinical meaning, namely,increases in calcium serum levels that are manifested in a subject bythe following side effects, depression of central and peripheral nervoussystem, muscular weakness, constipation, abdominal pain, lack ofappetite and, depressed relaxation of the heart during diastole.Symptomatic manifestations of hypercalcemia are triggered by astimulation of at least one of the following activities, intestinalcalcium transport, bone calcium metabolism and osteocalcin synthesis(reviewed in Boullion, R. et al. (1995) Endocrinology Reviews 16(2):200-257).

[0128] Compounds exhibiting reduced hypercalcemic activity can be testedin vivo or in vitro using methods known in the art and reviewed byBoullion, R. et al. (1995) Endocrinology Reviews 16(2): 200-257. Forexample, the serum calcium levels following administration of a vitaminD3 compounds of formula I or II can be tested by routine experimentation(Lemire, J. M. (1994) Endocrinology 135(6):2818-2821). Briefly, vitaminD3 compounds of formulas I and II can be administered intramuscularly tovitamin D₃-deficient subjects, e.g., rodents, e.g. mouse, or avianspecies, e.g. chick. At appropriate time intervals, serum calcium levelsand extent of calcium uptake can be used to determine the level of bonecalcium mobilization (BCM) and intestinal calcium absorption (ICA)induced by the tested vitamin D₃ compound as described in Norman, A. W.et at. (1993) .J Biol. Chem. 268(27):20022-20029. Compounds which uponaddition fail to increase the concentration of calcium in the bloodserum, thus showing decreased BCM and ICA responses compared to theirisomeric counterparts, are considered to have reduced hypercalcemicactivity. Compounds which have reduced toxicity compared to theirisomeric counterparts are considered to have reduced toxicity.Additional calcium homeostasis-related assays are described below in theCalcium and Phosphate Homeostasis section.

[0129] Hyperproliferative Conditions

[0130] In another aspect the present invention provides a method oftreating in a subject, a disorder characterized by aberrant activity ofa vitamin D3-responsive cell. The method involves administering to thesubject an effective amount of a pharmaceutical composition of a vitaminD3 compound of formula I or II such that the activity of the cell ismodulated. As used herein, the language “modulate” refers to increasesor decreases in the activity of a cell in response to exposure to acompound of the invention, e.g., the inhibition of proliferation and/orinduction of differentiation of at least a sub-population of cells in ananimal such that a desired end result is achieved, e.g. a therapeuticresult. In preferred embodiments, this phrase is intended to includehyperactive conditions that result in pathological disorders.

[0131] In certain embodiments, the cells to be treated arehyperproliferative cells. As described in greater detail below, thevitamin D3 compounds of formulas I and II can be used to inhibit theproliferation of a variety of hyperplastic and neoplastic tissues. Inaccordance with the present invention, vitamin D3 compounds of formulasI and II can be used in the treatment of both pathologic andnon-pathologic proliferative conditions characterized by unwanted growthof vitamin D3-responsive cells, e.g., hyperproliferative skin cells,immune cells, and tissue having transformed cells, e.g., such ascarcinomas, sarcomas and leukemias. In other embodiments, the cells tobe treated are aberrant secretory cells, e.g., parathyroid cells, immunecells.

[0132] As used herein, the terms “hyperproliferative” and “neoplastic”are used interchangeably, and include those cells having the capacityfor autonomous growth, i.e., an abnormal state or conditioncharacterized by rapidly proliferating cell growth. Hyperproliferativeand neoplastic disease states may be categorized as pathologic, i.e.,characterizing or constituting a disease state, or may be categorized asnon-pathologic, i.e., a deviation from normal but not associated with adisease state. The term is meant to include all types of cancerousgrowths or oncogenic processes, metastatic tissues or malignantlytransformed cells, tissues, or organs, irrespective of histopathologictype or stage of invasiveness. “Pathologic hyperproliferative” cellsoccur in disease states characterized by malignant tumor growth.Examples of non-pathologic hyperproliferative cells includeproliferation of cells associated with wound repair.

[0133] The use of vitamin D3 compounds of formulas I and II in treatinghyperproliferative conditions has been limited because of theirhypercalcemic effects. Thus, vitamin D3 compounds of formula I and IIcan provide a less toxic alternative to current methods of treatment.

[0134] In one embodiment, this invention features a method forinhibiting the proliferation and/or inducing the differentiation of ahyperproliferative skin cell, e.g., an epidermal or an epithelial cell,e.g. a keratinocytes, by contacting the cells with a vitamin D3 compoundof formula I or II. In general, the method includes a step of contactinga pathological or non-pathological hyperproliferative cell with aneffective amount of such vitamin D3 compound to promote thedifferentiation of the hyperproliferative cells The present method canbe performed on cells in culture, e.g. in vitro or ex vivo, or can beperformed on cells present in an animal subject, e.g., as part of an invivo therapeutic protocol. The therapeutic regimen can be carried out ona human or any other animal subject.

[0135] The vitamin D3 compounds of the present invention can be used totreat a hyperproliferative skin disorder. Exemplary disorders include,but are not limited to, psoriasis, basal cell carcinoma, keratinizationdisorders and keratosis. Additional examples of these disorders includeeczema; lupus associated skin lesions; psoriatic arthritis; rheumatoidarthritis that involves hyperproliferation and inflammation ofepithelial-related cells lining the joint capsule; dermatitides such asseborrheic dermatitis and solar dermatitis; keratoses such as seborrheickeratosis, senile keratosis, actinic keratosis. photo-induced keratosis,and keratosis follicularis; acne vulgaris; keloids and prophylaxisagainst keloid formation; nevi; warts including verruca, condyloma orcondyloma acuminatum, and human papilloma viral (HPV) infections such asvenereal warts; leukoplakia; lichen planus; and keratitis.

[0136] In an illustrative example, vitamin D3 compounds of formulas Iand II can be used to inhibit the hyperproliferation of keratinocytes intreating diseases such as psoriasis by administering an effective amountof these compounds to a subject in need of treatment. The term“psoriasis” is intended to have its medical meaning, namely, a diseasewhich afflicts primarily the skin and produces raised, thickened,scaling, nonscarring lesions. The lesions are usually sharply demarcatederythematous papules covered with overlapping shiny scales. The scalesare typically silvery or slightly opalescent. Involvement of the nailsfrequently occurs resulting in pitting, separation of the nail,thickening and discoloration. Psoriasis is sometimes associated witharthritis, and it may be crippling. Hyperproliferation of keratinocytesis a key feature of psoriatic epidermal hyperplasia along with epidermalinflammation and reduced differentiation of keratinocytes. Multiplemechanisms have been invoked to explain the keratinocytehyperproliferation that characterizes psoriasis. Disordered cellularimmunity has also been implicated in the pathogenesis of psoriasis.

[0137] Pharmaceutical compositions of vitamin D3 compounds of formulas Iand II can be delivered or administered topically or by transdermalpatches for treating dermal psoriasis. Alternatively, oraladministration is used. Additionally, the compositions can be deliveredparenterally, especially for treatment of arthritis, such as psoriaticarthritis, and for direct injection of skin lesions. Parenteral therapyis typically intra-dermal, intra-articular, intramuscular orintravenous. A preferred way to practice the invention is to apply thevitamin D3 compounds of formulas I and II, in a cream or oil basedcarrier, directly to the psoriatic lesions. Typically, the concentrationof the vitamin D3 compound in a cream or oil is 1-2%. Alternatively, anaerosol can be used topically. These compounds can also be orallyadministered.

[0138] In general, the route of administration is topical (includingadministration to the eye, scalp, and mucous membranes), oral, orparenteral. Topical administration is preferred in treatment of skinlesions, including lesions of the scalp, lesions of the cornea(keratitis), and lesions of mucous membranes where such directapplication is practical. Shampoo formulations are sometimesadvantageous for treating scalp lesions such as seborrheic dermatitisand psoriasis of the scalp. Mouthwash and oral paste formulations can beadvantageous for mucous membrane lesions, such as oral lesions andleukoplakia. Oral administration is a preferred alternative fortreatment of skin lesions and other lesions discussed above where directtopical application is not as practical, and it is a preferred route forother applications.

[0139] Intra-articular injection is a preferred alternative in the caseof treating one or only a few (such as 2-6) joints. Additionally, thetherapeutic compounds are injected directly into lesions (intra-lesionadministration) in appropriate cases. Intra-dermal administration is analternative for dermal lesions such as those of psoriasis.

[0140] The amount of the pharmaceutical composition to be administeredvaries depending upon the type of the disease of a patient, the severityof the disease, the type of the active vitamin D₃ compound of Formulas Ior II, among others. For example, the vitamin D3 compound of formula Ior II can be administered topically for treating hyperproliferative skinconditions at a dose in the range of 1 to 1000 μg per gram of topicalformulation.

[0141] Neoplasia

[0142] Another embodiment features methods for inhibiting theproliferation and/or reversing the transformed phenotype of vitaminD3-responsive hyperproliferative cells by contacting the cells with avitamin D3 compound of formula I or II. In general, the method includesa step of contacting pathological or non-pathological hyperproliferativecells with an effective amount of a vitamin D3 compound of formula I orII for promoting the differentiation of the hyperproliferative cells.The present method can be performed on cells in culture, e.g., in vitroor ex vivo, or can be performed on cells present in an animal subject,e.g., as part of an in vivo therapeutic protocol. The therapeuticregimen can be carried out on a human or other animal subject.

[0143] The terms “antineoplastic agent” and “antiproliferative agent”are used interchangeably herein and includes agents that have thefunctional property of inhibiting the proliferation of a vitaminD3-responsive cells, e.g., inhibit the development or progression of aneoplasm having such a characteristic, particularly a hematopoieticneoplasm.

[0144] As used herein, a “therapeutically effective anti-neoplasticamount” of a vitamin D3 compound of formula I or II refers to an amountof an agent which is effective, upon single or multiple doseadministration to the patient, in inhibiting the growth of a neoplasticvitamin D3-responsive cells, or in prolonging the survivability of thepatient with such neoplastic cells beyond that expected in the absenceof such treatment. As used herein, “inhibiting the growth” of theneoplasm includes the slowing, interrupting, arresting or stopping itsgrowth and metastases and does not necessarily indicate a totalelimination of the neoplastic growth.

[0145] As used herein, “a prophylactically effective anti-neoplasticamount” of a compound refers to an amount of a vitamin D3 compound offormula I or II which is effective, upon single or multiple doseadministration to the patient, in preventing or delaying the occurrenceof the onset of a neoplastic disease state.

[0146] The common medical meaning of the term “neoplasia” refers to “newcell growth” that results as a loss of responsiveness to normal growthcontrols, e.g. to neoplastic cell growth. A “hyperplasia” refers tocells undergoing an abnormally high rate of growth. However, as usedherein, the terms neoplasia and hyperplasia can be used interchangably,as their context will reveal, referring to generally to cellsexperiencing abnormal cell growth rates. Neoplasias and hyperplasiasinclude “tumors,” which may be either benign, premalignant or malignant.

[0147] The vitamin D3 compounds of formulas I and II can be testedinitially in vitro for their inhibitory effects in the proliferation ofneoplastic cells. Examples of cell lines that can be used aretransformed cells, e.g., the human promyeloid leukemia cell line HL-60,and the human myeloid leukemia U-937 cell line (Abe E. et al. (1981)Proc. Natl. Acad. Sci. USA 78:4990-4994; Song L. N. and Cheng T. (1992)Biochem Pharmacol 43:2292-2295; Zhou J. Y. et al. (1989) Blood 74:82-93;U.S. Pat. No. 5,401,733, U.S. Pat. No. 5,087,619). Alternatively, theantitumoral effects of vitamin D3 compounds of formulas 1 and 11 can betested in vivo using various animal models known in the art andsummarized in Bouillon, R. et al. (1995) Endocrine Reviews 16(2):233(Table E), which is incorporated by reference herein. For example, SLmice are routinely used in the art to test vitamin D3 compounds offormulas I and II as models for MI myeloid leukemia (Honma et al. (1983)Cell Biol. 80:201-204; Kasukabe T. et al. (1987) Cancer Res.47:567-572); breast cancer studies can be performed in, for example,nude mice models for human MX1 (ER) (Abe J. et al. (1991) Endocrinology129:832-837; other cancers, e.g., colon cancer, melanoma osteosarcoma,can be characterized in, for example, nude mice models as describe in(Eisman J. A. et al. (1987) Cancer Res. 47:21-25; Kawaura A. et al.(1990) Cancer Lett 55:149-152; Belleli A. (1992) Carcinogenesis13:2293-2298; Tsuchiya H. et al. (1993) J. Orthopaed Res. 11:122-130).

[0148] The subject method may also be used to inhibit the proliferationof hyperplastic/neoplastic cells of hematopoietic origin, e.g., arisingfrom myeloid, lymphoid or erythroid lineages, or precursor cellsthereof. For instance, the present invention contemplates the treatmentof various myeloid disorders including, but not limited to, acutepromyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronicmyelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. inOncol./Hemotol. 11:267-97). Lymphoid malignancies which may be treatedby the subject method include, but are not limited to acutelymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineageALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL),hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM).Additional forms of malignant lymphomas contemplated by the treatmentmethod of the present invention include, but are not limited tonon-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas,adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL),large granular lymphocytic leukemia (LGF) and Hodgkin's disease.

[0149] The term “leukemia” is intended to have its clinical meaning,namely, a neoplastic disease in which white corpuscle maturation isarrested at a primitive stage of cell development. The disease ischaracterized by an increased number of leukemic blast cells in the bonemarrow, and by varying degrees of failure to produce normalhematopoietic cells. The condition may be either acute or chronic.Leukemias are further typically categorized as being either lymphocytici.e., being characterized by cells which have properties in common withnormal lymphocytes, or myelocytic (or myelogenous), i.e., characterizedby cells having some characteristics of normal granulocytic cells. Acutelymphocytic leukemia (“ALL”) arises in lymphoid tissue, and ordinarilyfirst manifests its presence in bone marrow. Acute myelocytic leukemia(“AML”) arises from bone marrow hematopoietic stem cells or theirprogeny. The term acute myelocytic leukemia subsumes several subtypes ofleukemia: myeloblastic leukemia, promyelocytic leukemia, andmyelomonocytic leukemia. In addition, leukemias with erythroid ormegakaryocytic properties are considered myelogenous leukemias as well.

[0150] As used herein the term “leukemic cancer” refers to all cancersor neoplasias of the hemopoietic and immune systems (blood and lymphaticsystem). The acute and chronic leukemias, together with the other typesof tumors of the blood, bone marrow cells (myelomas), and lymph tissue(lymphomas), cause about 10% of all cancer deaths and about 50% of allcancer deaths in children and adults less than 30 years old. Chronicmyelogenous leukemia (CML), also known as chronic granulocytic leukemia(CGL), is a neoplastic disorder of the hematopoietic stem cell. The term“leukemia” is art recognized and refers to a progressive, malignantdisease of the blood-forming organs, marked by distorted proliferationand development of leukocytes and their precursors in the blood and bonemarrow.

[0151] In certain embodiments, the vitamin D3 compounds of formulas Iand II can be used in combinatorial therapy with conventional cancerchemotherapeutics. Conventional treatment regimens for leukemia and forother tumors include radiation, drugs, or a combination of both. Inaddition to radiation, the following drugs, usually in combinations witheach other, are often used to treat acute leukemias: vincristine,prednisone, methotrexate, mercaptopurine, cyclophosphamide, andcytarabine. In chronic leukemia, for example, busulfan, melphalan, andchlorambucil can be used in combination. All of the conventionalanti-cancer drugs are highly toxic and tend to make patients quite illwhile undergoing treatment. Vigorous therapy is based on the premisethat unless every leukemic cell is destroyed, the residual cells willmultiply and cause a relapse.

[0152] The subject method can also be useful in treating malignancies ofthe various organ systems, such as affecting lung, breast, lymphoid,gastrointestinal, and genito-urinary tract as well as adenocarcinomaswhich include malignancies such as most colon cancers, renal-cellcarcinoma, prostate cancer and/or testicular tumors, non-small cellcarcinoma of the lung, cancer of the small intestine and cancer of theesophagus.

[0153] The term “carcinoma” is art recognized and refers to malignanciesof epithelial or endocrine tissues including respiratory systemcarcinomas, gastrointestinal system carcinomas, genitourinary systemcarcinomas, testicular carcinomas, breast carcinomas, prostaticcarcinomas, endocrine system carcinomas, and melanomas. Exemplarycarcinomas include those forming from tissue of the cervix, lung,prostate, breast, head and neck, colon and ovary. The term also includescarcinosarcomas, e.g., which include malignant tumors composed ofcarcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to acarcinoma derived from glandular tissue or in which the tumor cells formrecognizable glandular structures.

[0154] The term “sarcoma” is art recognized and refers to malignanttumors of mesenchymal derivation.

[0155] According to the general paradigm of vitamin D3 involvement indifferentiation of transformed cells, exemplary solid tumors that can betreated according to the method of the present invention include vitaminD3-responsive phenotypes of sarcomas and carcinomas such as, but notlimited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, and retinoblastoma.

[0156] Determination of a therapeutically effective anti-neoplasticamount or a prophylactically effective anti-neoplastic amount of thevitamin D3 compound of formula I or II, can be readily made by thephysician or veterinarian (the “attending clinician”), as one skilled inthe art, by the use of known techniques and by observing resultsobtained under analogous circumstances. The dosages may be varieddepending upon the requirements of the patient in the judgment of theattending clinician, the severity of the condition being treated and theparticular compound being employed. In determining the therapeuticallyeffective antineoplastic amount or dose, and the prophylacticallyeffective antineoplastic amount or dose, a number of factors areconsidered by the attending clinician, including, but not limited to:the specific hyperplastic/neoplastic cell involved; pharmacodynamiccharacteristics of the particular agent and its mode and route ofadministration; the desirder time course of treatment; the species ofmammal; its size, age, and general health; the specific diseaseinvolved; the degree of or involvement or the severity of the disease;the response of the individual patient; the particular compoundadministered; the mode of administration; the bioavailabilitycharacteristics of the preparation administered; the dose regimenselected; the kind of concurrent treatment (i.e., the interaction of thevitamin D3 compounds of formulas I and II with other co-administeredtherapeutics); and other relevant circumstances. U.S. Pat. No.5,427,916, for example, describes method for predicting theeffectiveness of antineoplastic therapy in individual patients, andillustrates certain methods which can be used in conjunction with thetreatment protocols of the instant invention.

[0157] Treatment can be initiated with smaller dosages which are lessthan the optimum dose of the compound. Thereafter, the dosage should beincreased by small increments until the optimum effect under thecircumstances is reached. For convenience, the total daily dosage may bedivided and administered in portions during the day if desired. Atherapeutically effective antineoplastic amount and a prophylacticallyeffective anti-neoplastic amount of a vitamin D3 compound of formula Ior II is expected to vary from about 0.1 milligram per kilogram of bodyweight per day (mg/kg/day) to about 100 mg/kg/day.

[0158] Compounds which are determined to be effective for the preventionor treatment of tumors in animals, e.g., dogs, rodents, may also beuseful in treatment of tumors in humans. Those skilled in the art oftreating tumor in humans will know, based upon the data obtained inanimal studies, the dosage and route of administration of the compoundto humans. In general, the dosage and route of administration in humansis expected to be similar to that in animals.

[0159] The identification of those patients who are in need ofprophylactic treatment for hyperplastic/neoplastic disease states iswell within the ability and knowledge of one skilled in the art. Certainof the methods for identification of patients which are at risk ofdeveloping neoplastic disease states which can be treated by the subjectmethod are appreciated in the medical arts, such as family history ofthe development of a particular disease state and the presence of riskfactors associated with the development of that disease state in thesubject patient. The present application also describes other prognostictests which can be used to make, or to augment a clinical predicationabout the use of the method of the present invention. A clinicianskilled in the art can readily identify such candidate patients, by theuse of, for example, clinical tests, physical examination andmedical/family history.

[0160] Immunomodulatory Effects

[0161] In another aspect, this invention provides a method formodulating the activity of an immune cell by contacting the cell with avitamin D3 compound of formulas I or II. Vitamin D3 compounds are knownin the art for their inhibitory effects on the antigen-specific immunesystem. As used herein, the phrase “inhibition of an immune response” isintended to include decreases in T cell proliferation and activity,e.g., a decrease in IL₂, interferon-γ, GM-CSF synthesis and secretion(Lemire, J. M. (1992) J. Cell Biochemistry 49:26-31, Lemire, J. M. etal. (1994) Endocrinology 135 (6): 2813-2821; Bouillon, R. et al. (1995)Endocine Review 16 (2):231-32)

[0162] In one embodiment, the present invention provides a method forsuppressing immune activity in an immune cell by contacting apathological or non-pathological immune cell with an effective amount ofa vitamin D3 compound of formulas I or II to thereby inhibit an immuneresponse relative to the cell in the absence of the treatment. Thepresent method can be performed on cells in culture, e.g., in vitro orex vivo, or can be performed on cells present in an animal subject,e.g., as part of an in vivo therapeutic protocol. In vivo treatment canbe carried out on a human or other animal subject.

[0163] The vitamin D3 compound of formula I or II can be testedinitially in vitro for their inhibitory effects on T cell proliferationand secretory activity, as described in Reichel, H. et al., (1987) Proc.Natl. Acad. Sci. USA 84:3385-3389; Lemire, J. M. et al. (1985) J.Immunol 34:2032-2035. Alternatively, the immunosuppressive effects canbe tested in vivo using the various animal models known in the art andsummarized by Bouillon, R. et al. (1995) Endocine Reviews 16(2) 232(Tables 6 and 7). For examples, animal models for autoimmune disorders,e.g., lupus, thyroiditis, encephalitis, diabetes and nephritis aredescribed in (Lemire J. M. (1992) J. Cell Biochem. 49:26-31; Koizumi T.et al. (1985) Int. Arch. Allergy Appl. Immunol. 77:396-404; Abe J. etal. (1990) Calcium Regulation and Bone Metabolism 146-151; Fournier C.et al. (1990) Clin. Immunol Immunopathol. 54:53-63; Lemire J. M. andArcher D. C. (1991) J. Clin. Invest 87:1103-1107); Lemire, J. M. et al.,(1994) Endocrinology 135 (6):2818-2821; Inaba M. et al. (1992)Metabolism 41:631-635; Mathieu C. et al. (1992) Diabetes 41:1491-1495;Mathieu C. et al. (1994) Diabetologia 37:552-558; Lillevang S. T. et al.(1992) Clin. Exp. Immunol. 88:301-306, among others). Models forcharacterizing immunosuppressuve activity during organ transplantation,e.g., skin graft, cardiac graft, islet graft, are described in JordanS.C. et al. (1988) v Herrath D (eds) Molecular, Cellular and ClinicalEndocrinology 346-347; Veyron P. et al. (1993) Transplant Immunol.1:72-76; Jordan S.C. (1988) v Herrath D (eds) Molecular, Cellular andClinical Endocrinology 334-335; Lemire J. M. et al. (1992)Transplantation 54:762-763; Mathieu C. et al. (1994) Transplant Proc.26:3128-3129).

[0164] After identifying certain test compounds as effective suppresorsof an immune response in vitro, these compounds can be used in vivo aspart of a therapeutic protocol. Accordingly, another embodiment providesa method of suppressing an immune response, comprising administering toa subject a pharmaceutical preparation of a vitamin D3 compound offormula I or II, so as to inhibit immune reactions such as graftrejection, autoimmune disorders and inflammation.

[0165] For example, the subject vitamin D3 compounds of formulas I andII can be used to inhibit responses in clinical situations where it isdesirable to downmodulate T cell responses. For example, ingraft-versus-host disease, cases of transplantation, autoimmune diseases(including, for example, diabetes mellitus, arthritis (includingrheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis,psoriatic arthritis), multiple sclerosis, encephalomyelitis, diabetes,myasthenia gravis, systemic lupus erythematosis, autoimmune thyroiditis,dermatitis (including atopic dermatitis and eczematous dermatitis),psoriasis, Sjögren's Syndrome, including keratoconjunctivitis siccasecondary to Sjögren's Syndrome, alopecia areata, allergic responses dueto arthropod bite reactions, Crohn's disease, aphthous ulcer, iritis,conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma,allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis,proctitis, drug eruptions, leprosy reversal reactions, erythema nodosumleprosum, autoimmune uveitis, allergic encephalomyelitis, acutenecrotizing hemorrhagic encephalopathy, idiopathic bilateral progressivesensorineural hearing loss, aplastic anemia, pure red cell anemia,idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis,chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue,lichen planus, Crohn's disease, Graves ophthalmopathy, sarcoidosis,primary biliary cirrhosis, uveitis posterior, and interstitial lungfibrosis). Downmodulation of immune activity will also be desirable incases of allergy such as, atopic allergy.

[0166] As described before, determination of a therapeutically effectiveimmunosuppressive amount can be readily made by the attending clinician,as one skilled in the art, by the use of known techniques and byobserving results obtained under analogous circumstances. Compoundswhich are determined to be effective in animals, e.g., dogs, rodents,may be extrapolated accordingly to humans by those skilled in the art.Starting dose/regimen used in animals can be estimated based on priorstudies. For example, doses of vitamin D3 compounds of formulas I and IIto treat autoimmune disorders in rodents can be initially estimated inthe range of 0.1 g/kg/day to 1 g/kg/day, administered orally or byinjection.

[0167] Those skilled in the art will know based upon the data obtainedin animal studies, the dosage and route of administration in humans isexpected to be similar to that in animals. Exemplary dose ranges to beused in humans are from 0.25 to 10 μg/day, preferably 0.5 to 5 μg/dayper adult (U.S. Pat. No. 4,341,774).

[0168] Calcium and Phosphate Homeostasis

[0169] The present invention also relates to a method of treating in asubject a disorder characterized by deregulation of calcium metabolism.This method comprises contacting a pathological or non-pathologicalvitamin D3 responsive cell with an effective amount of a vitamin D3compound of formula I or II to thereby directly or indirectly modulatecalcium and phosphate homeostasis. The term “homeostasis” isart-recognized to mean maintenance of static, or constant, conditions inan internal environment. As used herein, the term “calcium and phospatehomeostasis” refers to the careful balance of calcium and phosphateconcentrations, intracellularly and extracellularly, triggered byfluctuations in the calcium and phosphate concentration in a cell, atissue, an organ or a system. Fluctuations in calcium levels that resultfrom direct or indirect responses to vitamin D3 compounds of formulas Iand II are intended to be included by these terms. Techniques fordetecting calcium fluctuation in vivo or in vitro are known in the art.

[0170] Exemplary Ca⁺⁺ homeostasis related assays include assays thatfocus on the intestine where intestinal ⁴⁵Ca²⁺ absorption is determinedeither 1) in vivo (Hibberd K. A. and Norman A. W. (1969) Biochem.Pharmacol. 18:2347-2355; Hurwitz S. et al. (1967) J. Nutr. 91:319-323;Bickle D. D. et al. (1984) Endocrinology 114:260-267), or 2) in vitrowith everted duodenal sacs (Schachter D. et al. (1961) Am. J. Physiol200:1263-1271), or 3) on the genomic induction of calbindin-D_(28k) inthe chick or of calbindin-D_(9k) in the rat (Thomasset M. et al. (1981)FEBS Lett. 127:13-16; Brehier A. and Thomasset M. (1990) Endocrinology127:580-587). The bone-oriented assays include: 1) assessment of boneresorption as determined via the release of Ca²⁺ from bone in vivo (inanimals fed a zero Ca²⁺ diet) (Hibberd K. A. and Norman A. W. (1969)Biochem. Pharmacol. 18:2347-2355; Hurwitz S. et al. (1967) J. Nutr.91:319-323), or from bone explants in vitro (Bouillon R. et al. (1992)J. Biol. Chem. 267:3044-3051), 2) measurement of serum osteocalcinlevels [osteocalcin is an osteoblast-specific protein that after itssynthesis is largely incorporated into the bone matrix, but partiallyreleased into the circulation (or tissue culture medium) and thusrepresents a good market of bone formation or turnover] (Bouillon R. etal. (1992) Clin. Chem. 38:2055-2060), or 3) bone ash content (Norman A.W. and Wong R. G. (1972) J. Nutr. 102:1709-1718). Only onekidney-oriented assay has been employed. In this assay, urinary Ca²⁺excretion is determined (Hartenbower D. L. et al. (1977) Walter deGruyter, Berlin pp 587-589); this assay is dependent upon elevations inthe serum Ca²⁺ level and may reflect bone Ca²⁺ mobilizing activity morethan renal effects. Finally, there is a “soft tissue calcification”assay that has been employed to detect the consequences of 1α,25(OH)₂D3or analog-induced severe hypercalcemia. In this assay a rat isadministered an intraperitoneal dose of ⁴⁵Ca²⁺, followed by seven dailyrelative high doses of 1α,25(OH)₂D3 or the analog of interest; in theevent of onset of a severe hypercalcemia, soft tissue calcification canbe assessed by determination of the ⁴⁵Ca²⁺ level. In all these assays,vitamin D3 compounds or formulas I and II are administered to vitaminD-sufficient or -deficient animals, as a single dose or chronically(depending upon the assay protocol), at an appropriate time intervalbefore the end point of the assay is quantified.

[0171] In certain embodiments, vitamin D3 compounds of formulas I and IIcan be used to modulate bone metabolism. The language “bone metabolism”is intended to include direct or indirect effects in the formation ordegeneration of bone structures, e.g., bone formation, bone resorption,etc., which may ultimately affect the concentrations in serum of calciumand phosphate. This term is also intended to include effects of vitaminD3 compounds in bone cells, e.g. osteoclasts and osteoblasts, that mayin turn result in bone formation and degeneration. For example, it isknown in the art, that vitamin D3 compounds of formulas I and II exerteffects on the bone forming cells, the osteoblasts through genomic andnon-genomic pathways (Walters M. R. et al. (1982) J. Biol. Chem.257:7481-7484; Jurutka P. W. et al. (1993) Biochemistry 32:8184-8192;Mellon W. S. and DeLuca H. F. (1980) J. Biol. Chem. 255:4081-4086).Similarly, vitamin D3 compounds of formulas I and II are known in theart to support different activities of bone resorbing osteoclasts suchas the stimulation of differentiation of monocytes and mononuclearphagocytes into osteoclasts (Abe E. et al. (1988) J. Bone Miner Res.3:635-645; Takahashi N. et al. (1988) Endocrinology 123:1504-1510;Udagawa N. et al. (1990) Proc. Natl. Acad. Sci. USA 87:7260-7264).Accordingly, vitamin D3 compounds of formulas I and II that modulate theproduction of bone cells can influence bone formation and degeneration.

[0172] The present invention provides a method for modulating bone cellmetabolism by contacting a pathological or a non-pathological bone cellwith an effective amount of a vitamin D3 compound of formula I or II tothereby modulate bone formation and degeneration. The present method canbe performed on cells in culture, e.g., in vitro or ex vivo, or can beperformed in cells present in an animal subject, e.g., cells in vivo.Exemplary culture systems that can be used include osteoblast celllines, e.g., ROS 17/2.8 cell line, monocytes, bone marrow culture system(Suda T. et al. (1990) Med. Res. Rev. 7:333-366; Suda T. et al. (1992)J. Cell Biochem. 49:53-58) among others. Selected compounds can befurther tested in vivo, for example, animal models of osteopetrosis andin human disease (Shapira F. (1993) Clin. Orthop. 294:34-44).

[0173] In a preferred embodiment, a method for treating osteoporosis isprovided, comprising administering to a subject a pharmaceuticalpreparation of a vitamin D3 compound of formula I or II to therebyameliorate the condition relative to an untreated subject. The rationalefor utilizing vitamin D3 compounds in the treatment of osteoporosis issupported by studies indicating a decrease in serum concentration of1α,25(OH)₂D3 in elderly subjects (Lidor C. et al. (1993) Calcif . TissueInt. 52:146-148). In vivo studies using vitamin D3 compounds in animalmodels and humans are described in Bouillon, et al. (1995) EndocrineReviews 16(2):229-231.

[0174] Vitamin D3 compounds of formulas I and II can be tested inovarectomized animals, e.g., dogs, rodents, to assess the changes inbone mass and bone formation rates in both normal and estrogen-deficientanimals. Clinical trials can be conducted in humans by attendingclinicians to determine therapeutically effective amounts of the vitaminD3 compounds of formulas I and II in preventing and treatingosteoporosis.

[0175] Preferred compounds to be tested include 3-epi forms of 3-epiforms of 1α (OH)D₃ as shown in Example II, which shows the production of1α (QH)-3-epi-D₃ in the rat osteosarcoma cell line UMR-106. The 3 epiconversion of 1α (OH)-D₃ presents the possibility of a yet improved ofthis compound.

[0176] In other embodiments, therapeutic applications of the vitamin D3compounds of formulas I and II include treatment of other diseasescharacterized by metabolic calcium and phosphate deficiencies. Exemplaryof such diseases are the following: osteoporosis, osteodystrophy,osteomalacia, rickets, osteitis fibrosa cystica, renal osteodystrophy,osteosclerosis, anti-convulsant treatment, osteopenia,fibrogenesis-imperfecta ossium, secondary hyperparathyrodism,hypoparathyroidism, hyperparathyroidism, cirrhosis, obstructivejaundice, drug induced metabolism, medullary carcinoma, chronic renaldisease, hypophosphatemic VDRR, vitamin D-dependent rickets,sarcoidosis, glucocorticoid antagonism, malabsorption syndrome,steatorrhea, tropical sprue, idiopathic hypercalcemia and milk fever.

[0177] Hormone Secretion

[0178] In yet another aspect, the present invention provides a methodfor modulating hormone secretion of a vitamin D3-responsive cell, e.g.,an endocrine cell. The language “hormone secretion” is art-recognizedand includes both genomic and non-genomic activities of vitamin D3compounds of formulas I and II that control the transcription andprocessing responsible for secretion of a given hormone e.g.,parathyroid hormone (PTH), calcitonin, insulin, prolactin (PRL) and TRHin a vitamin D3 responsive cell (Bouillon, R. et al. (1995) EndocrineReviews 16(2):235-237). The language “vitamin D3 responsive cells” asused herein is intended to include endocrine cells which respond tovitamin D3 compounds of formulas I and II by altering gene expressionand/or post-transcriptional processing secretion of a hormone. Exemplaryendocrine cells include parathyroid cells, pancreatic cells, pituitarycells, among others.

[0179] The present method can be performed on cells in culture, e.g. invitro or ex vivo, or on cells present in an animal subject, e.g., invivo. Vitamin D3 compounds of formulas I and II can be initially testedin vitro using primary cultures of parathyroid cells. Other systems thatcan be used include the testing by prolactin secretion in rat pituitarytumor cells, e.g., GH4C1 cell line (Wark J. D. and Tashjian Jr. A. H.(1982) Endocrinology 111:1755-1757; Wark J. D. and Tashjian Jr. A. H.(1983) J. Biol. Chem. 258:2118-2121; Wark J. D. and Gurtler V. (1986)Biochem. J. 233:513-518) and TRH secretion in GH4C1 cells.Alternatively, the effects of vitamin D3 compounds of formulas I and IIcan be characterized in vivo using animals models as described in Nko M.et al. (1982) Miner Electrolyte Metab. 5:67-75; Oberg F. et al. (1993)J. Immunol. 150:3487-3495; Bar-Shavit Z. et al. (1986) Endocrinology118:679-686; Testa U. et al. (1993) J. Immunol. 150:2418-2430; NakamakiT. et al. (1992) Anticancer Res. 12:1331-1337; Weinberg J. B. andLarrick J. W. (1987) Blood 70:994-1002; Chambaut-Guérin A. M. andThomopoulos P. (1991) Eur. Cytokine New. 2:355; Yoshida M. et al. (1992)Anticancer Res. 12:1947-1952; Momparler R. L. et al. (1993) Leukemia7:17-20; Eisman J. A. (1994) Kanis J A (eds) Bone and Mineral Research2:45-76; Veyron P. et al. (1993) Transplant Immunol. 1:72-76; Gross M.et al. (1986) J Bone Miner Res. 1:457-467; Costa E. M. et al. (1985)Endocrinology 117:2203-2210; Koga M. et al. (1988) Cancer Res.48:2734-2739; Franceschi R. T. et al. (1994) J. Cell Physiol.123:401-409; Cross H. S. et al. (1993) Naunyn Schmiedebergs Arch.Pharmacol. 347:105-110; Zhao X. and Feldman D. (1993) Endocrinology132:1808-1814; Skowronski R. J. et al. (1993) Endocrinology132:1952-1960; Henry H. L. and Norman A. W. (1975) Biochem. Biophys.Res. Commun. 62:781-788; Wecksler W. R. et al. (1980) Arch. Biochem.Biophys. 201:95-103; Brumbaugh P. F. et al. (1975) Am. J. Physiol.238:384-388; Oldham S. B. et al. (1979) Endocrinology 104:248-254;Chertow B. S. et al. (1975) J. Clin Invest. 56:668-678; Canterbury J. M.et al. (1978) J. Clin. Invest. 61:1375-1383; Quesad J. M. et al. (1992)J. Clin. Endocrinol. Metab. 75:494-501.

[0180] In certain embodiments, the vitamin D3 compounds of the presentinvention can be used to inhibit parathyroid hormone (PTH) processing,e.g., transcriptional, translational processing, and/or secretion of aparathyroid cell as part of a therapeutic protocol. Therapeutic methodsusing these compounds can be readily applied to all diseases, involvingdirect or indirect effects of PTH activity, e.g., primary or secondaryresponses. For example, it is known in the art that PTH induces theformation of 1,25-dihydroxy vitamin D3 in the kidneys, which in turn inincreases calcium and phosphate absorption from the intestine thatcauses hypercalcemia. Thus inhibition of PTH processing and/or secretionwould indirectly inhibit all of the responses mediated by PTH in vivo.Accordingly, therapeutic applications for these vitamin D3 compounds offormulas I and II include treating diseases such as secondaryhyperparathyroidism of chronic renal failure (Slatopolsky E. et al.(1990) Kidney Int. 38:S41-S47; Brown A. J. et al. (1989) J. Clin.Invest. 84:728-732). Determination of therapeutically affective amountsand dose regimen can be performed by the skilled artisan using the datadescribed in the art.

[0181] Protection Against Neuronal Loss

[0182] In yet another aspect, the present invention provides a method ofprotecting against neuronal loss by contacting a vitamin D3 responsivecell, e.g., a neuronal cell, with a vitamin D3 compound of formula I orII to prevent or retard neuron loss. The language “protecting against”is intended to include prevention, retardation, and/or termination ofdeterioration, impairment, or death of a neurons. The language “vitaminD3 responsive cells” as used herein is intended to include neuronalcells which respond to vitamin D3 compounds of formulas I and II byaltering gene expression and/or intracellular metabolism. Exemplaryneuronal cells include hippocampal cells, dopaminergic cells,cholinergic cells, among others.

[0183] Neuron loss can be the result of any condition of a neuron inwhich its normal function is compromised. Neuron deterioration can bethe result of any condition which compromises neuron function which islikely to lead to neuron loss. Neuron function can be compromised by,for example, altered biochemistry, physiology, or anatomy of a neuron.Deterioration of a neuron may include membrane, dendritic, or synapticchanges which are detrimental to normal neuronal functioning. The causeof the neuron deterioration, impairment, and/or death may be unknown.Alternatively, it may be the result of age- and/or disease-relatedchanges which occur in the nervous system of a subject.

[0184] When neuron loss is described herein as “age-related”, it isintended to include neuron loss resulting from known and unknown bodilychanges of a subject which are associated with aging. When neuron lossis described herein as “disease-related”, it is intended to includeneuron loss resulting from known and unknown bodily changes of a subjectwhich are associated with disease. It should be understood, however,that these terms are not mutually exclusive and that, in fact, manyconditions that result in the loss of neurons are both age- anddisease-related.

[0185] Exemplary age-related diseases associated with neuron loss andchanges in neuronal morphology include, for example, Alzheimer'sDisease, Pick's Disease, Parkinson's Disease, Vascular Disease,Huntington's Disease, and Age-Associated Memory Impairment. InAlzheimer's Disease patients, neuron loss is most notable in thehippocampus, frontal, parietal, and anterior temporal cortices,amygdala, and the olfactory system. The most prominently affected zonesof the hippocampus include the CA1 region, the subiculum, and theentorhinal cortex. Memory loss is considered the earliest and mostrepresentative cognitive change because the hippocampus is well known toplay a crucial role in memory. Pick's Disease is characterized by severeneuronal degeneration in the neocortex of the frontal and anteriortemporal lobes which is sometimes accompanied by death of neurons in thestriatum. Parkinson's Disease can be identified by the loss of neuronsin the substantia nigra and the locus ceruleus. Huntington's Disease ischaracterized by degeneration of the intrastriatal and corticalcholinergic neurons and GABA-ergic neurons. Parkinson's and Huntington'sDiseases are usually associated with movement disorders, but often showcognitive impairment (memory loss) as well.

[0186] Age-Associated Memory Impairment (AAMI) is another age-associateddisorder that is characterized by memory loss in healthy, elderlyindividuals in the later decades of life. Crook, T. et al. (1986) Devel.Neuropsych. 2(4):261-276. Presently, the neural basis for AAMI has notbeen precisely defined. However, neuron death with aging has beenreported to occur in many species in brain regions implicated in memory,including cortex, hippocampus, amygdala, basal ganglia, cholinergicbasal forebrain, locus ceruleus, raphe nuclei, and cerebellum. Crook, T.et al. (1986) Devel. Neuropsych. 2(4):261-276.

[0187] Vitamin D3 compounds of formulas I and II can protect againstneuron loss by genomic or non-genomic mechanisms. Nuclear vitamin D3receptors are well known to exist in the periphery but have also beenfound in the brain, particularly in the hippocampus and neocortex.Non-genomic mechanisms may also prevent or retard neuron loss byregulating intraneuronal and/or peripheral calcium and phosphate levels.Furthermore, vitamin D3 compounds of formulas I and II may protectagainst neuronal loss by acting indirectly, e.g., by modulating serumPTH levels. For example, a positive correlation has been demonstratedbetween serum PTH levels and cognitive decline in Alzheimer's Disease.

[0188] The present method can be performed on cells in culture, e.g. invitro or ex vivo, or on cells present in an animal subject, e.g., invivo. Vitamin D3 compounds of formulas I and II can be initially testedin vitro using neurons from embryonic rodent pups (See e.g. U.S. Pat.No. 5,179,109-fetal rat tissue culture), or other mammalian (See e.g.U.S. Pat. No. 5,089,517-fetal mouse tissue culture) or non-mammaliananimal models. These culture systems have been used to characterize theprotection of peripheral, as well as, central nervous system neurons inanimal or tissue culture models of ischemia, stroke, trauma, nervecrush, Alzheimer's Disease, Pick's Disease, and Parkinson's Disease,among others. Examples of in vitro systems to study the prevention ofdestruction of neocortical neurons include using in vitro cultures offetal mouse neurons and glial cells previously exposed to variousglutamate agonists, such as kainate, NMDA, andα-amino-3-hydroxy-5-methyl-4-isoxazolepronate (AMPA). U.S. Pat. No.5,089,517. See also U.S. Pat. No. 5,170,109 (treatment of ratcortical/hippocampal neuron cultures with glutamate prior to treatmentwith neuroprotective compound); U.S. Pat. Nos. 5,163,196 and 5,196,421(neuroprotective excitatory amino acid receptor antagonists inhibitglycine, kainate, AMPA receptor binding in rats).

[0189] Alternatively, the effects of vitamin D3 compounds of formulas Iand II can be characterized in vivo using animals models. Neurondeterioration in these model systems is often induced by experimentaltrauma or intervention (e.g. application of toxins, nerve crush,interruption of oxygen supply). For example, in order to demonstratethat certain N-methyl-D-aspartate (NMDA), an excitatory amino acidneurotransmitter receptor, antagonists were useful as anticonvulsantsand neuroprotectants, the inventors in U.S. Pat. No. 4,957,909 employeda model wherein Swiss-albino mice and rat hippocampal neurons weresubjected to overstimulation of excitatory amino acid receptorssubsequent to treatment with the NMDA antagonists. A similar study wasperformed wherein the utility of certain NMDA antagonists as agents thatprevent neurodegeneration was demonstrated by treating mice with NMDAsubsequent to treatment with the NMDA antagonists. U.S. Pat. No.5,168,103.

[0190] Smooth Muscle Cells

[0191] In yet another aspect, the present invention provides a method ofmodulating the activity of a vascular smooth muscle cell by contacting avitamin D3-responsive smooth muscle cell with a vitamin D3 compounds offormulas I or II to activate or, preferably, inhibit the activity of thecell. The language “activity of a smooth muscle cell” is intended toinclude any activity of a smooth muscle cell, such as proliferation,migration, adhesion and/or metabolism.

[0192] In certain embodiments, the vitamin D3 compounds of formulas Iand II can be used to treat diseases and conditions associated withaberrant activity of a vitamin D3-responsive smooth muscle cell. Forexample, the present invention can be used in the treatment ofhyperproliferative vascular diseases, such as hypertension inducedvascular remodeling, vascular restenosis and atherosclerosis. In otherembodiments, the present invention can be used in treating disorderscharacterized by aberrant metabolism of a vitamin D3-responsive smoothmuscle cell, e.g., arterial hypertension.

[0193] The present method can be performed on cells in culture, e.g. invitro or ex vivo, or on cells present in an animal subject, e.g., invivo. Vitamin D3 compounds of formulas I and II can be initially testedin vitro as described in Catellot et al. (1982), J. Biol. Chem. 257(19):11256.

[0194] This invention is further illustrated by the following exampleswhich in no way should be construed as being further limiting. It isunderstood by the ordinary skilled artisan that production of a vitaminD3 compound of formula I or II in a cell is indicative that suchcompound is biologically active in such cell, and thus that it can beused in treating conditions arising from aberrant activity of suchcells. For example, production of vitamin D3 compounds of formulas I andII in keratinocytes is indicative that such vitamin D3 compounds arebiologically active in those cells and can be used in treatingconditions such as psoriasis. The contents of all cited references(including literature references, issued patents, published patentapplications, and co-pending patent applications) cited throughout thisapplication are hereby expressly incorporated by reference.

EXAMPLES EXAMPLE I Isolation and Identification of a Cyclic EtherMetabolite of 1α,25-dihydroxy-vitamin D₃ in Human Keratinocytes

[0195] As described herein, 1α25(OH)₂-3-epi-D₃ is metabolized into aless polar metabolite than 1α25(OH)₂-3-epi-D₃, peak M1, in humankeratinocytes (FIG. 2). FIG. 2 shows the HPLC profile and UV spectra ofthe metabolites produced in human keratinocytes incubated with1α25(OH)₂-3-epi3D₃ (1 uM) for 24H. On a straight phase HPLC system, thismetabolite (M1) is more polar than 25 (OH) D₃ but less polar than1α25(OH)₂D₃ and similar to that of 1α(OH)D₃. Mass spectrometric analysisreveals a molecular ion of 414 m/z, which is 2 mass units less than thestarting 1α25(OH)₂D₃, shown in FIG. 3. FIG. 3 shows the mass spectra of1α (OH)₂-3-epi-D₃ (M) (upper panel) and its cyclic ether metabolite (M₁)(lower panel) isolated from human keratinocytes incubated with1α25(OH)₂-3-epi-D₃(1 uM) for 24 h. The typical fragments at m/z 134 and152 m/z indicate an unmodified A-ring and cistriene structure. A doublebond introduced at either C, D rings or the side chain would beconsistent with the molecular weight. However, this type of unsaturatedmetabolite still possesses a free 25-hydroxyl group and is expected tohave similar retention time as the starting compound; this iscontradicting to what was observed. Furthermore, the absence of massfragments at 59 m/z suggests the absence of a 25-hydroxyl group. Theabsence of the familiar side chain cleavage fragments at 251, 269 and287 m/z also suggests a modified 25-hydroxyl group and a possiblestructural change at C-20 to retard the cleavage at carbons 17 and 20. Acyclic structure as shown in FIGS. 3 and 4 is supported by these massspectrometric and chromatographic evidences. This proposed structure isconsistent with the loss of m/z 58 (acetone) to form m/z 356 and thesubsequent fragments at 338, 320 and 314.

[0196] It is probable that the 3-epi modification of the A-ring allowsalternate side chain reactions to occur. FIG. 4 summarizes the proposedmetabolic pathway for the formulation of the cyclic ether metabolite of1α25(OH)₂-3-epi-D₃. The formation of a cyclic ether structure couldresult from a hydroxylation at either C-17 or C-20 and the subsequentreaction with the 25-hydroxyl group to form an ether linkage as shown inFIG. 4. This type of metabolic reactions are known to occur inhydroxylated fatty acids. Thus, it is probable that some of theunidentified metabolites can be C-17 or C-20-hydroxylated metabolites of1α25(OH)₂-3-epi-D₃.

EXAMPLE II Isolation and Identification of a 3-Epi Metabolite of 1αhydroxy-vitamin D₃ in Human Keratinocytes

[0197]FIG. 5A shows the metabolism of 1α(OH)-D₃ into its 3 epi form inthe osteosarcoma cell line UMR-106. Peak A represents the 3-epi form of1α(OH)D₃. Peak B corresponds to the substrate, 1α(OH)D₃. The insertpanels show the UV spectra of the various metabolites as monitored byphotodiode array detector. FIG. 5B shows a schematic representation ofthe 3-epimerization of 1α(OH)D₃ into 1α(OH)-3-epi vitamin D₃. Similar to1α(OH)D₃, 1α(OH)-3-epi vitamin D₃ can be converted into the25-hydroxylated form in vivo.

[0198] 1α(OH)D₃ compounds are currently used in the treatment ofosteoporosis. Thus, 3-epi forms of these compounds may be used assubstitutes for 1α(OH)D₃ compounds in treating osteoporosis.

EXAMPLE III Confirmation of 3-epi Configuration of 1α(OH) 3-epi vitaminD₃

[0199] To confirm the production of 1α(OH) 3-epi vitamin D₃ in bonecells. the metabolites of 1α-3-epi-D₃ produced by the osteosarcoma cellline (UMR-106) were analyzed by mass spectroscopy. FIG. 6 shows the massspectra of 1α(OH)D₃ (upper panel) and its 3-epi metabolite (lowerpanel). A comparison of these two mass spectra revealed difference inpeaks observed only in the 3-epi metabolite, for example, fragmentshaving molecular weights of approximately m/z 57, 217, 312 and 529(lower panel). The mass spectrum of the 3-epi metabolite wasindependently confirmed to be 1α(OH) 3-epi vitamin D₃.

EXAMPLE IV Enhanced Stability In Vivo of 1α(OH) 3-epi Vitamin D₃Compared to Its Isomeric Counterpart

[0200] The stability of 1α(OH) 3-epi vitamin D₃ in vivo wascharacterized by monitoring the changes in the concentration of 1α(OH)3-epi vitamin D₃ and its isomeric counterpart at various time intervals.In particular, FIG. 7 shows the HPLC profile and UV spectra of themetabolites produced in rat osteosarcoma cell lines (UMR-106) which wereincubated with 1α(OH)D₃ for 24, 48, or 84 hours. Peak M and S representthe relative concentrations of 1α(OH) 3-epi vitamin D₃ and its isomericcounterpart, respectively, at the time intervals tested. The persistentduration of peak M relative to peak S after 48 and 84 hour-incubationsindicates that 3-epi metabolite of 1α(OH)D₃ are more stable in vivo thanits isomeric counterpart

[0201] Equivalents

[0202] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents of thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

What is claimed is:
 1. An isolated cyclic ether vitamin D3 compoundhaving the formula (I) as follows:

,wherein A₁, A₂ and A₃ are a single or a double bond; X, R₁, R₂, R₃, R₄and R₅ are selected from the group consisting of a hydrogen, a halogen,a haloalkyl, a hydroxy, a hydroxy-protecting group, an alkyl, analkenyl, an alkynyl, an alkoxy, an aryl group and a heterocyclic group.2. An isolated 3-epi form of 1α-hydroxy-vitamin D3 compounds having theformula II as follows:

, wherein A₁ is a single, a double, or a triple bond; A₂, A₃ and A₄ areeach independently selected from the group consisting of a single or adouble bond; R₂, R₃, R₄, R₇, R₈ and R₉ are independently selected fromthe group consisting of a hydrogen, a deuterium, a deuteroalkyl, ahydroxy, an alkyl, an alkoxide, an O-acyl, a halogen, a haloalkyl, ahydroxyalkyl, an arnine or a thiol group, and wherein the pairs of R₂and R₃, and R₄ and R₇ taken together are an oxygen atom; and R₅ and R₆are independently selected from the group consisting of a hydrogen, adeuterium, a halogen, an alkyl, a hydroxyalkyl, a haloalkyl, and adeuteroalkyl.
 3. The compound of claim 2, which is 1α(OH) vitamin D3,1α,24 dihydroxy 3-epi vitamin D₃, 1α hydroxy 24-ethyl 3-epi vitamin D₃,1α hydroxy 24-methyl 3-epi vitamin D₃, or 1α,24-dihydroxy 24-methyl3-epi vitamin D₃.
 4. A method of treating a disorder characterized by anaberrant activity of a vitamin D₃-responsive cell, comprisingadministering to a subject an effective amount of a vitamin D₃ compoundhaving the formula (I) or (II) of any of claims 1 or 2, such that theaberrant activity of the vitamin D₃-responsive cell is reduced.
 5. Themethod of claim 4, wherein the disorder comprises an aberrant activityof a hyperproliferative skin cell.
 6. The method of claim 4, wherein thedisorder comprises an aberrant activity of an endocrine cell.
 7. Themethod of claim 6, wherein the endocrine cell is a parathyroid cell andthe aberrant activity is processing and/or secretion of parathyroidhormone.
 8. The method of claim 7, wherein the disorder is secondaryhyperparathyroidism.
 9. The method of claim 8, wherein the disordercomprises an aberrant activity of a bone cell.
 10. The method of claim9, wherein the disorder is selected from the group consisiting ofosteoporosis, osteodystrophy, senile osteoporosis, osteomalacia,rickets, osteitis fibrosa cystica, renal osteodystrophy, secondaryhyperparathyrodism, cirrhosis, and chronic renal disease.
 11. The methodof claim 4, wherein the subject is a mammal.
 12. The method of claim 11,wherein the mammal is a human.
 13. A method of ameliorating aderegulation of calcium and phosphate metabolism, comprisingadministering to a subject a therapeutically effective amount of a 3-epivitamin D3 compound of any of claims 2 or 3, so as to ameliorate thederegulation of the calcium and phosphate metabolism.
 14. The method ofclaim 13, wherein the deregulation of the calcium and phosphatemetabolism leads to osteoporosis.
 15. A pharmaceutical compositioncomprising, a therapeutically effective amount of a vitamin D₃ compoundof any of claims 1 or 2, and a pharmaceutically acceptable carrier. 16.The composition of claim 15, which is suitable for topical or oraladministration.
 17. A packaged compound, comprising a vitamin D₃compound of any of claims 1 or 2, packaged with instructions for use ofthe compound for treating a disorder characterized by an aberrantactivity of a vitamin D₃-responsive cell.